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Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis
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  • Marek Polák
    Marek Polák
    Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czech Republic
    Department of Biochemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic
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  • Jiří Černý
    Jiří Černý
    Laboratory of Structural Bioinformatics of Proteins, Institute of Biotechnology of the Czech Academy of Sciences, 14220 Prague, Czech Republic
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    Orcidhttps://orcid.org/0000-0002-1969-9304
  • Petr Novák*
    Petr Novák
    Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czech Republic
    Department of Biochemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic
    *Email: [email protected]. Fax: +420 241 062 156. Tel: +420 325 873 610.
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    Orcidhttps://orcid.org/0000-0001-8688-529X
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Analytical Chemistry

Cite this: Anal. Chem. 2024, 96, 4, 1478–1487
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https://doi.org/10.1021/acs.analchem.3c03759
Published January 16, 2024

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Abstract

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Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein–DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

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Structural proteomics has considerably advanced the field of structural and molecular biology in recent years by enabling us to address questions related to the structure and dynamics of proteins and their complexes. Methods of structural proteomics are particularly useful for studying transcription factor–DNA interactions (1) such as those involved in transcription. Transcription is primarily regulated by transcription factors (TFs), proteins that specifically activate or inhibit this process by forming complexes with DNA. For instance, Forkhead transcription factor “O” 4 (FOXO4) is known to bind core motif 5′-(C/A)(C/C)AAA(C/T)A-3′ (Insulin Response Element) and further activate transcription that includes energy metabolism control. (2,3) Yet, despite extensive research in this area, our understanding of transcription regulation and interaction between TFs and their binding motifs remains limited. To better understand these processes, we must further clarify the structural mechanism underlying the formation and interaction of protein–DNA complexes. (4−6)
Protein–DNA complexes have been studied using cross-linking reactive probes, (7−9) hydrogen–deuterium exchange (HDX) (7,8,10−12) and radical covalent labeling. (1) It was demonstrated that surface mapping of biomolecules, detected by high-resolution MS analysis, offers useful information related to the protein structure or its interaction with other biomolecules. (1)
Various radical probes are currently available, with diverse reactivity toward different amino acids, (13−18) but the most commonly used labeling chemistry consists of modification by hydroxyl radicals. (19) Among the methods involving hydroxyl radicals, the most promising approach was introduced by Hambly and Gross in 2005 and is referred to as fast photochemical oxidation of proteins (FPOP). (20)
In FPOP, proteins are irreversibly labeled in a quench-flow capillary system by a single hit of hydroxyl radical, generated from hydrogen peroxide by an excimer laser. This reaction is immediately quenched by a suitable quenching reagent in the flow system. The rationale of this approach lies in the preferential oxidation of solvent-accessible side chains of the investigated molecule, and thus mapping of the protein structure. (21,22) Hydroxyl radicals promote modification of 14 of 20 naturally occurring amino acids. The most reactive are the sulfur-containing amino acids, namely, cysteine and methionine, followed by the residues of the aromatic amino acids, namely, phenylalanine, tyrosine, histidine, and tryptophan. (23−26) As such, FPOP has been applied to various studies, including mapping of protein conformational changes, protein–protein interactions, (27) the structure and topology of membrane proteins, (28−31) and large biomolecules (32) such as antibodies. (20,27−31,33,34)
Although bottom-up MS analysis has long been a method of choice for studying FPOP modified samples, top-down protocols have recently demonstrated their potential for analyzing singly oxidized proteoforms. (1,35,36) Originally, a top-down technology was introduced to determine not only biomolecule sequences but also post-translational and other (bio)chemical modifications of biomolecules. (37−39) One of the advantages of analyzing samples by top-down MS is the precise determination of the molecular mass of proteins and other proteoforms. However, when the molecular mass of the analyzed species exceeds ∼1 kDa, the monoisotopic peak ceases to be the most abundant signal in the spectrum of the analyzed protein or peptide. Mass spectra of multiply charged proteins and fragment ions over ∼10 kDa do not produce observable monoisotopic peaks anymore, so the ion signal is dispersed into several, commonly overlapping isotopic peaks. As a result, the MS/MS spectra are complex and show a low signal-to-noise ratio (SNR). (40,41)
Precisely designed to overcome these difficulties, the technique of protein isotope depletion was introduced in 1997. (42) Protein isotope depletion relies on incubating bacteria in media with depleted heavy isotopes, e.g., carbon and nitrogen. Several studies have described the benefits of isotope depletion for analyzing proteins up to ∼20 kDa, (43−45) but Gallagher et al. stood out for using protein isotope depletion to improve the resolution of MS/MS spectra and thus fragment ion assignment. (46) More recently, Popovic et al. applied protein depletion to analyze the proteome of cells by 21T-FT-ICR mass spectrometry. (47) In common, these studies leveraged the potential of protein depletion for improving the accuracy of mass determination and sequence coverage of biomolecules by MS.
Considering the above, this study aimed at exploring the concept of protein isotope depletion and its advantages for increasing the spatial resolution of top-down MS analysis of FPOP samples. To this end, we prepared an isotopically depleted version of FOXO4-DBD and oxidized this sample under two different conditions, that is, with and without its DNA binding element, IRE. First, we used a bottom-up MS approach to analyze isotopically natural (IN) protein samples to acquire single-amino acid information. Subsequently, we applied a top-down MS approach to analyze an isotopically depleted (ID) version of the protein, which resulted in enhanced sequence coverage and more precise assignment of labeled residues. ID fragmentation yielded new ions in the MS/MS spectra, which were not detected in the IN spectra. The additional fragment ions offered more detailed information about the solvent-accessible FOXO4-DBD surface, thus enabling ab initio design of a FOXO4-IRE structural model.

Experimental Section

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Materials and Chemicals

All solvents (LC/MS grade) and chemicals were purchased from Merck (Germany) unless stated otherwise.

Protein Expression of Isotopically Natural (IN) FOXO4-DBD

The expression was performed in Terrific-Broth (TB) medium. All details about the expression of IN-FOXO4-DBD can be found in the Supporting Information.

Protein Expression of Isotopically Depleted (ID) FOXO4-DBD

The expression of FOXO4-DBD was performed in M9 minimal medium containing glucose (99.95% of 12C, Merck) and ammonium sulfate (NH4SO4, 99.99% of 14N, Merck) as a source of carbon and nitrogen, respectively. All details about the expression of ID-FOXO4-DBD can be found in the Supporting Information.

Protein Purification

Both IN- and ID-FOXO4-DBD were purified in the same fashion, as described in detail in the Supporting Information.

FOXO4·IRE Complex Formation

Forward (5′-GAC TAT CAA AAC AAC GC-3′) and reverse (5′-GCG TTG TTT TGA TAG TC-3′) complementary strands, whose sequence was retrieved from a previous study, (19) were obtained from IDT (Coralville, USA) in an HPLC quality. A stock solution of dsIRE was prepared by mixing both strands in an equimolar ratio in LC-MS water. The mixture was incubated at 90 °C for 3 min and cooled to room temperature to form dsIRE. The 30 μM and 35 μM samples of IN/ID FOXO4-DBD and dsIRE, respectively, were mixed to form a complex and ensure that all protein was bonded in complex with DNA. The complex was diluted in 150 mM ammonium acetate, pH 6.8, and incubated at room temperature for 15 min to obtain IN/ID-FOXO4·IRE complex at 30 μM final concentration.

Fast Photochemical Oxidation of Proteins (FPOP)

The FPOP labeling was performed in an in-house built quench-flow reactor as described previously. (1) Prior to the FPOP experiment, glutamine (10 mM) was added to the protein samples. Briefly, ID/IN FOXO4-DBD (30 μM) with and without dsIRE (35 μM), in 150 mM ammonium acetate, pH 6.8, was continuously mixed in a T-mixer with H2O2 (15 mM during reaction). The mixture was irradiated by an excimer laser (COMPex 50 KrF, Coherent Inc., USA). A mixture of the sample and H2O2 was subjected to a single shot at a wavelength of 248 nm, frequency 15 Hz, energy 107 mJ, 20 ns pulse duration, and 2.24 mJ/cm2 radiant exposure. The exclusion volume was 16%. The reaction was quenched by immediate mixing with 75 mM methionine. The samples were collected in an Eppendorf tube containing 3000 U of Catalase (Merck, USA).

Top-Down MS Detection

Protein–DNA samples were denatured by adding 4 M urea and 1 μM MgCl2; the mixture was incubated at a bench for 15 min. DNA was digested by adding 1 μL of benzonase endonuclease (Merck) and incubated at 30 °C for an additional 15 min. The mixture was then loaded onto a reverse-phase microtrap column (Optimize technologies, USA), desalted using 0.1% FA, and eluted with 80% ACN, 0.1% FA. The desalted protein was further diluted 5 times using 30% ACN, 0.1% FA solution, and sprayed using a nESI source in positive mode at 120 °C desolvation temperature. MS and MS/MS analyses were performed on a solariX XR mass spectrometer equipped with 15 T magnet (Bruker Daltonics, Billerica, USA), which was externally calibrated using the sodium trifluoroacetate to achieve 1 ppm mass accuracy. The time-of-flight was set to 1.1 ms, with the collision energy ranging between −3.0 to −5.0 V, and data were acquired with 2 M data point transient starting at 200 amu. At first, MS intact spectra were acquired in a broadband mode (m/z 200–2500) by accumulating ions for 0.1 s–0.2 s and collecting 128 scans.
For MS/MS, singly oxidized protein ions of three charge states (+14, +13, +12) were isolated using a multiCASI (multicontinuous accumulation of selected ions) in a quadrupole and then transferred to ICR cell for electron-capture dissociation (ECD). The single oxidized ions of IN-FOXO4 were isolated at 844.50, 909.30, and 984.90 amu, and the isolation window was ±1.0 amu. ID-FOXO4-DBD singly oxidized ions were isolated at nominal values 843.93, 908.77, and 984.42 amu, and the isolation window was ±0.6 amu. An ion-accumulation from 3.0 to 5.0 s was tuned to reach the intensity of the precursor ion image current of ∼108 prior to the ECD experiment. The ECD was done by setting the parameters to obtain optimal fragmentation as follows: ECD pulse length 0.065–0.075 s, bias 0.90–1.0 V, and lens 14.0–15.0 V. The hollow cathode current was 1.5 A. Control spectra of unmodified ions were acquired using the same condition as the oxidized ones. Data were acquired by collecting 128 scans in a technical triplicate for both apo and holo forms.

Top-Down Data Processing

Raw data were processed using Data Analysis 5.3 (Bruker Daltonics, Billerica, USA), MS2Links software, (27) and in-house built software. Details related to top-down data processing are included in the Supporting Information.

Bottom-Up LCMS Detection

Samples for bottom-up analysis were digested using Trypsin/LysC (Promega, USA) and LysC (Promega, USA). Respective protease was added at a protease:protein ratio 1:40 (m:m) and incubated overnight at 37 °C. Additional protease (m/m 1:20) was added after overnight incubation for another 6 h. IRE was digested by adding bensonase endonuclease (250 U, Merck) to the sample for 30 min at 37 °C. Digestion was terminated by addition of trifluoroacetic acid (TFA, 0.1%).
LC was performed to separate the peptides as described previously, (1) albeit with one minor modification. An LC run consisted of a 35 min linear gradient of 2–35% solvent B. LC was directly hyphenated to a trapped ion mobility-quadrupole time-of-flight mass spectrometer (timsTOF Pro, Bruker Daltonics) for MS/MS analysis. MS/MS analysis was performed as described previously. (1)
LC-MS analysis was performed on a timsTOF Pro mass spectrometer. MS analysis was performed using the same method as MSMS, but without collisional dissociation and fragment accumulation.
Data were processed using a PeaksX+ Software (Bioinformatic Solutions Inc., Waterloo, ON, Canada) against a FOXO4-DBD sequence as described previously. (1,48) Peptide intensities were extracted from LC-MS trace using Data Analysis 5.3 (Bruker Daltonics, USA) for all observed charge states, quantified, and statistically analyzed, as described previously. (49) The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (50) partner repository with the data set identifier PXD044447.

Homology Modeling

The homology model of the FOXO4/IRE complex was based on the available crystal structure 3l2c, (51) extending the 3l2c template DNA sequence “–CTATGTAAACAAC–” to the IRE “GACTATCAAAACAACGC” sequence. The necessary nucleotide substitutions as well as residues missing at the 5′ and 3′ termini were modeled using the MMB program. (52,53) The backbone conformation of the newly built termini was set to the canonical B form DNA using dinucleotide conformation class (NtC) BB00. (54,55) The geometry of the initial model was further equilibrated during a 200 ns molecular dynamics simulation in GROMACS 2021.4 (56) using the ff14SB (57) force field for FOXO4 and the tumuc1 force field (58) for DNA. The model was placed in a rectangular box with the 10 Å shortest distance from the walls. The box was filled with TIP3P model water, and Na+ and Cl– ions were added to reach a charge-neutral system with 100 mM salt concentration. The system was simulated with noncovalent cutoffs of 10 Å at 300 K and 1 bar with the V-rescale modified Berendsen thermostat and the Parrinello–Rahman barostat.

Results and Discussion

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Top-down MS analysis of FPOP enables us to determine the protein–DNA interaction interface between FOXO4 and dsDNA (namely DAF16), as shown in our recent study. (1) However, the complex spectra and lower sequence coverage prevent us from reaching single-amino acid resolution, as in the bottom-up MS approach. To increase fragment intensity and to reduce the complexity of top-down MS spectra, we prepared IN and 13C/15N-doubly depleted (ID) versions of FOXO4-DBD to investigate the benefit of isotopic depletion for studying the interaction interface between FOXO4 and IRE by top-down MS analysis. We therefore expressed and purified the DNA-binding domain of FOXO4 at a length of 82–1863 (numbering according to the FOXO4 wild-type sequence) containing two (G–2S–1) additional residues located in the N-terminus. So as to simplify the top-down MS data interpretation, we further used the 1 to 107 common numbering of c and z fragment ions. The conversion of fragment ion numbering to the wild-type FOXO4 sequence is shown in Figure S1.
Initially, TB and M9 media were used to recombinantly express IN and ID protein, respectively (Methods in the Supporting Information). Both proteins were purified by using the same protocol. The intact protein analysis revealed the homogeneity of both protein samples (Figure S2) and confirmed the isotopic depletion of FOXO4-DBD (Figure 1A,D), while electrophoretic mobility shift assay (EMSA, Methods in the Supporting Information) demonstrated the ability of both proteins to bind dsIRE (Figure S3). Subsequently, the ID and IN forms of FOXO4-DBD were oxidized with and without dsIRE using FPOP. Figure 1B,C shows multiple oxidized proteoforms of IN-FOXO4-DBD with and without dsIRE, respectively, after FPOP. Similarly, Figure 1E,F displays multiple oxidized proteoforms of ID-FOXO4-DBD with and without dsIRE, respectively. With dsIRE (Figure 1C,F), the proteoforms were less oxidized than in solution alone (Figure 1B,E). These results confirm the protection of residues directly or indirectly involved in the protein–DNA interaction.

Figure 1

Figure 1. Zoomed mass spectrum on a +14-charge state (m/z 842–851) showing an isotopic distribution of isotopically natural (IN-, A) and isotopically depleted (ID-, D) FOXO4-DBD. Fast photochemical oxidation of IN-FOXO4 without (B) and with (C) dsIRE. Fast photochemical oxidation of ID-FOXO4 without (E) and with (F) dsIRE.

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Given the confirmation of the interaction between FOXO4-DBD and IRE by EMSA (Figure S3) and by intact MS analysis (Figure 1B,C,E,F), the investigation proceeded with top-down MS analysis. In order to demonstrate the advantage of isotopic depletion for analyzing singly oxidized proteins, singly oxidized proteoforms of IN and ID samples were isolated in the quadrupole and fragmented by ECD in the ICR cell (Figure S4). (35) By combining multiCASI simultaneous isolation of three charge states with ECD fragmentation, we were able to detect 101 nonoxidized fragment ions (57 c ions, 44 z ions) when analyzing IN samples. However, only 57 fragment ions (30 c ions, 27 z ions; see Figure S5A) were intense enough for quantification, resulting in a sequence coverage of 27% (see Figure S5B). Under the same experimental conditions, three charge states were isolated and fragmented using the ID sample. The isotopic purity of the ID protein allowed us to isolate oxidized ions in the quadrupole with a narrower isolation window of ±1 and ±0.6 Da for IN and ID samples, respectively. In total, 148 fragment ions were then annotated and manually validated, including small ions, c3, c4, and z3, which did not show other observable isotopic peaks (Figures S6 and S7). Unlike in the IN sample, 95 fragment ions (54 c ions, 42 z ions; see FigureS8A) were intense enough for quantification, resulting in a sequence coverage of 45% (Figure S8B). Thus, another 24 c-ions and 15 z-ions were available for quantification in ID (Figure 2) compared to in IN samples.

Figure 2

Figure 2. Histograms displaying the number of quantified fragment ions generated by ECD fragmentation of singly oxidized precursor ions of IN-FOXO4-DBD and ID-FOXO4-DBD.

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ID MS/MS spectra displayed (i) lower complexity than IN spectra, which greatly reduced the overlap of isotopes/fragment ions, (ii) a monoisotopic peak for all fragment ions, and (iii) an increased signal-to-noise ratio (SNR) (Figure S9). To further illustrate these improvements, we can consider the m/z region 1056–1061, which shows the difference in the complexity of the ECD spectra between IN and ID oxidized proteins (Figure 3).

Figure 3

Figure 3. Zoomed ECD spectrum obtained upon fragmentation of isotopically natural (A) and isotopically depleted (B) FOXO4-DBD. The [c21]2+ is indicated with blue asterisks; the low-abundance [c57]6+ fragment ion is denoted by green squares; and its oxidized form, [c57+O]6+, is indicated by pink dots. The oxidized fragment ion [z38+O]4 is denoted by magenta triangles.

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Figure 3A clearly shows the overlap of three isotopes for two fragment ions, [c21]2+ and [c57]6+, thus precluding reliable quantification of the [c57]6+ fragment ion. Moreover, its oxidized form, [c57+O]6+, has a very low signal-to-noise ratio, which is close to the limit of detection. In contrast, fragment ion intensity is approximately three times higher in ID (Figure 3B) than in IN MS/MS spectra. Hence, the [c21]2+ fragment ion consists of only two peaks, and both [c57]6+ [c57+O]6+ fragments are now observed as intense ions in spectra, where the most abundant peak is the monoisotopic peak, which significantly exceeds the noise level.
During FPOP, the high reactivity of ·OH radicals promotes not only oxygen additions (+15.9949 Da) but also other modifications (Table S1). (59) For instance, the conversion to the keto form (60) (addition of +13.9793 Da) or lysine carbonylation (36,61) (loss of −1.0313 Da) can also be detected during the FPOP analysis. In a typical bottom-up data analysis, (62) where small peptides are created by enzymatic digestion and subsequently separated on a reversed-phase column, these modifications are easily detected by DDA analysis. However, these modifications are difficult to detect by top-down MS analysis for several reasons: (i) they are not major products of the reaction and thus are not observed at a higher intensity, and (ii) they may overlap with other reaction products in the MS/MS spectrum. (1,35)
By simplifying the mass spectra and improving the signal-to-noise ratio (Figure S9), isotopic depletion helps to overcome the aforementioned limitations. As a result, these modifications can be detected in top-down MS/MS spectra of ID samples, as exemplified by the [c73]8+ fragment ion (Figure 4). The control spectrum does not show any oxidized [c73+O]8+ fragment ion (Figure 4A, black). But an MS/MS spectrum of oxidized FOXO4-DBD without (Figure 4B,D; blue) and with the IRE (Figure 4C,E; red) provided an oxidized [c73+O]8+ fragment ion. Additionally, oxidation to a keto form (indicated by green dots) and protein carbonylation (indicated by a yellow dot) were also detected during MS analysis, albeit to a lesser extent with IRE. This result confirms both the wide reactivity of ·OH radicals toward different residues and the protection of some residues by IRE (Figure 4B,C). Panels D and E of Figure 4, conversely, both show that neither lysine carbonylation nor keto-oxidation is identified in the zoomed-in view of the MS/MS spectrum of IN-FOXO4-DBD.

Figure 4

Figure 4. MS/MS spectrum zoomed in the m/z range 1008–1012.300. The control ECD spectrum of unmodified ID-FOXO4-DBD is colored in black in the top panel (A). The ECD spectrum of oxidized ID-FOXO4-DBD with (B) and without IRE (C) is colored in blue and in red. The isotopic distribution of both [c73]8+ and [c73+O]8+fragment ions is denoted by transparent asterisks. Yellow dots denote lysine carbonylation within the protein, represented by the loss of 1.013 Da, while the green dots represent the oxidation of protein to its keto form (+13.9793). An ECD MS/MS spectrum of IN-FOXO4-DBD without IRE (D) and with IRE (E) shows no visible lysine carbonylation or oxidation to keto form.

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To obtain structural information based on the assignment of oxidized residues and the increasing intensity of fragment ions, the extent of oxidation was calculated for both the apo and holo forms. This allowed us to visualize the differences between vicinal fragment regions (Figure 5). The difference map for isotopically natural FOXO4 represents an example of lower sequence coverage (Figure 5A). In this case, only several residues might be assigned as oxidized based solely on a sequence coverage and on amino acid reactivity toward hydroxyl radicals. Thus, the overall extent of oxidation is a combination of the sum of extents of oxidations of residues located in each region and their exposure toward solvent. In contrast, fragmenting isotopically depleted protein provides increased signal-to-noise ratio, which resulted in elevated sequence coverage, and thus, more residues might be assigned as oxidized ones (Figure 5B). Analysis of the ID protein revealed changes in the oxidation patterns of several residues of FOXO4-DBD upon binding to IRE (Figure 5C). These were further visualized in an in silico model of FOXO4-IRE (Figure 6, Figure S10). The first detected oxidized residues, K10, N16 and W18, were protected upon the complex formation and deduced from c[10], c[16], and c[18] fragment ions. Residues N16 and W18 were found to be directly interacting with the IRE according to the structural model (Figure 6) and also according to the previous study. (51) This is also consistent with the z[87–92] region. Helix H1 (S22-A34) contains several residues that were protected by IRE, in particular residues Q21, Y23, L26/I27, and I31, which were covered by c[21], c[23], c[26–28], c[30–31], z[77–83], and z[84–86]. This is in agreement with the previously published HDX (8) and structural studies. (51,63) However, we have also observed a higher oxidation rate for Q29, E32, and P35 deduced from c[29], c[32], c[34–35], and z[73–76], located throughout the H1 helix and intervening loop far away from the protein–DNA interface (Figure 6, Figure S10). Residues in helices H2 and H4, namely, Y45, W47/M48, and Y54, (51) showed different oxidation patterns, with some being less oxidized and some being more oxidized upon protein binding to IRE. The residues were covered by regions c[43–46], c[47–49], z[62–65], c[54], and z[54]. Moreover, R50, oriented toward the solvent, (64) was found to be more oxidized in the presence of IRE (Figure 5C, Figure 6). (1,51) The protection of K58 and deprotection of D60 residue was deduced based on regions c[58], c[59–61], and z[47–49]. Oxidation of N62 is deduced from z[46], alongside the region c[62–65]/z[44–45] which pinpoints the oxidation to both S63 and S64 residues. One may hypothesize that both N62 and S63 are protected upon the complex formation, (3,51) while S64 residue is oriented more toward the solvent and thus might be more oxidized upon complex formation (Figure 6). (51) A helix H3 (G66-H78), the main contributor of interaction with the major groove of IRE, (51) was found to be heavily protected. Residues of helix H3, namely, W67, H73, and H78, are covered by regions c[66–68], c[72–73], c[77–79], z[33–36], and z[40–42] and display the direct protection of helix by the major groove of IRE. Next, there has been a multitude of residues oxidized on strand S2 and wing W1 (F81–S93) bearing different oxidation patterns. Residues K83 and S93, covered by z[25] and z[15], were both protected. This observations are supported by a structural model and complementary mutagenesis studies (3) demonstrating direct interaction of K83 and S93 residues with the DNA. Even though F81 is located next to I82 and also is more reactive, we pinpoint hotspot to the I82, because residue F81 is located away from the solvent and interacts with IRE in our model. Thus, residues I82, H85, E87, and S92, covered by regions z[26–28], z[22–23], z[21], and z[16], were detected as more oxidized.

Figure 5

Figure 5. Plots indicating changes in oxidation rates between apo and holo forms of isotopically natural FOXO4 (A) and isotopically depleted FOXO4 (B); assessed by ECD fragmentation in multiCASI mode (Figure S5, Figure S8). Blue histograms represent changes in which region/residue was protected by IRE, and red histograms represent changes which resulted in deprotection of region/residue by IRE. (C) Changes obtained in ID-FOXO4-DBD were visualized into the differential oxidation map of FOXO4-DBD. The bold sequence represents spatial resolution achieved by fragmentation of isotopically depleted FOXO4-DBD. Colored residues were also detected by bottom-up analysis, as shown in Figure S11B and Table S1.

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Figure 6

Figure 6. An in silico structural model of FOXO4-DBD·IRE (PDB template 3L2C) (22) with the highlighted differently oxidized regions/residues detected by both top-down analyses for natural version (A) or depleted version (B) of FOXO4-DBD. The individual residues detected in either bottom-up approach or deduced from top-down were highlighted in the model and colored. Blue: regions/residues detected as more modified in apo form; red: regions/residues detected as more modified in holo form.

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Finally, data from isotopically depleted samples allowed us to resolve the solvent accessibility of residues located at strand S3 at a single residue resolution. Residues W94, W95, and M96 are covered by regions z[12], z[13], z[14], c[92–94], and c[95–97]. Overall effect of IRE binding is the stabilization of strand S3 and deprotection and protection of W94 and W95 residues, respectively, as W94 interacts with IRE. (3,51,63) The analysis of smaller z ions led to the assignment of L97 and P99 as oxidized ones based on [z8] and [z10] fragment ions, respectively. Altogether, the isotopic depletion and selective gas-phase enrichment and fragmentation of oxidized ions lead to the detection of 30 residues, namely K10, N16, W18, Q21, Y23, L26/I27, Q29, I31, E32, P35, Y45, R50, K58, D60, N62, S63, S64, H73, H78, I82, K83, H85, E87, S92, S93, W94, W95, M96, L97, and P99. Out of them, 22 residues were deduced solely from the isotopically depleted top-down data set. The other 7 were also detected using the bottom-up analysis described below.

Bottom-Up Analysis

To assess the top-down MS data, the same isotopically natural samples were also subjected to a bottom-up analysis. The LysC and Trypsin/LysC digestion of IN samples yielded 22 peptides, providing a sequence coverage of over 96% (see Figure S11A). Across the sequence of FOXO4-DBD, 18 residues were modified (Figure S11B and Table S1). Nine residues, namely, W18, Y23, Y45, W47, M48, H73, H78, W94#, and W95, showed decreased oxidation in the complex with IRE, indicating protection. By contrast, residues R15, E46, Y54, W94, W94##, and E100 showed increased oxidation/modification levels upon complex formation. Five residues were not affected by IRE binding. The single-residue resolution of the bottom-up approach supported data from our ab initio model, in line with previously published mutagenesis studies, (3) which reported that most residues involved in the interaction with the major groove of DNA were less oxidized/modified.
The agreement between the bottom-up and top-down results demonstrates the usefulness of the top-down and complementarity of both techniques. For instance, residues W18, Y23, Y45, Y54, H73, H78, W94, W95, and M96 differed in the extent of their modification upon IRE binding in the bottom-up data set, as observed by top-down MS analysis. This confirms that top-down MS analysis is a reliable technique for detecting oxidation levels at different residues.
Notwithstanding the above, some differences were detected between the results of the two techniques: (i) Bottom-up workflow benefits from LC for resolving even isobaric/isomeric modifications prior to mass spectrometric detection, a feature that has been referred to as “sub amino acid resolution”. (34) As a case in point, oxidized W94 can have several forms depending on its position on the indole ring. This finding was also detected by the top-down workflow, albeit only as a cumulative modification, represented as a sum of individual extents and without indicating the exact position of the modification (Figure S12). (ii) Limited sequence coverage, especially for larger proteins, is a known weakness of top-down techniques in general. Thus, residue oxidation determined by bottom-up can be overlooked by top-down approaches due to the lack of usable fragment ions intense enough for detection, as in the case of W47/M48 residues, both of which were detected in bottom-up but not individually in the top-down spectra. (iii) Bottom-up analysis of all proteoforms present in the sample also detects modifications other than the +16 Da (22) due to oxidation resulting from enzymatic digestion, as shown by R15/R72 deguanidylation, E25, E46, and E100 decarboxylation, and H73 and H78 conversion into aspartate in our bottom-up data set (Figure S11, Table S1). In the top-down experiment, we performed targeted gas-phase accumulation of species with mass increased by +16 Da (indicative of oxidative modification) prior to fragmentation. For this reason, all other modifications were not identified in the top-down experiment. Nevertheless, this limitation could be easily circumvented by including these other theoretical modifications in the accumulation, if necessary. Despite requiring the identification of the species to be included, selective gas-phase accumulation is a great advantage of top-down analysis for significantly enriching the included forms.
In our experiments, this enrichment enabled the detection and assignment of 22 new oxidized residues that were not detected by bottom-up MS analysis, as discussed above. These oxidations were impossible to detect by a bottom-up workflow given the low limit of detection, which is especially limiting for forms with low levels of oxidation and concentrations in the mixture of trypsinated peptides. As such, the benefits of top-down MS analysis in improving the limit of detection can actually outweigh its other limitations.
One obstacle to the broader use of isotopic depletion in MS analysis is the availability of processing software. The current software portfolio is restricted to the natural occurring isotopic distribution, and the data deconvolution mainly relies on averagine function. (45) This function does not allow the deconvolution of an altered isotopic pattern. Nevertheless, this problem may also be solved in the near future by adopting ion deconvolution using measured collision cross sections of trapped ions. (65) For larger proteins, MSMS spectra are necessarily complex, regardless of using protein isotope depletion. However, for small and midsized proteins, (41) the advantage of isotopic depletion is significant.

Conclusion

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Protein isotope depletion improves the detection and quantification of FPOP oxidation by a top-down MS analysis. This approach has three main advantages: (i) more precisely isolating singly oxidized ions in quadrupole filter prior to fragmentation; (ii) enhancing the intensities of unmodified and oxidized fragment ions; and (iii) improving the resolution of fragment ions in MS/MS spectra by reducing the number of isotopic peaks and thus reducing the overlap of existing peaks, including those of individual isotopes.
Combining isotopic depletion with top-down analysis of FPOP samples boosts sequence coverage by 19% and identifies 22 more oxidized residues in comparison to bottom-up MS analysis. Nevertheless, bottom-up and top-down analyses show highly consistent results, demonstrating their complementarity. The more detailed information on the interaction interface obtained in this study enables the ab initio design of FOXO4·IRE complex formation. Even beyond interactions between transcription factors and DNA, the approach reported in this study holds great promise for future research of noncovalent interactions by top-down MS, particularly for more complex biomolecular assemblies, whose interaction dynamics often remains unclear in solution.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c03759.

  • Additional methods: Expression and purification of isotopically natural and depleted FOXO4-DBD, top-down data processing, electrophoretic mobility shift assay; Additional Figures: (Figure S1) FOXO4 sequence with both top-down and wild-type numbering, (Figure S2) ESI-MS spectra of desalted protein samples, (Figure S3) EMSA gel, (Figure S4) broadband ECD spectrum of IN-/ID-FOXO4-DBD, (Figure S5) quantified ions of IN-FOXO4-DBD, (Figure S6) zoom of [c4]1+ and [c5]1+ ions, (Figure S7) zoom of [z3]1+ and [z4]1+ ions, (Figure S8) quantified ions of ID-FOXO4-DBD, (Figure S9) zoom-in of ECD MSMS spectra of IN-/ID-FOXO4-DBD, (Figure S10) ab initio model of FOXO4-IRE with wild-type numbering, (Figure S11) bottom-up analysis of IN-FOXO4-DBD, (Figure S12) quantified extent of oxidation of W94 residue; Additional table: (Table S1) all modifications identified in bottom-up approach (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Marek Polák - Institute of Microbiology of the Czech Academy of Sciences, 14220 Prague, Czech Republic;  Department of Biochemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic
    • Jiří Černý - Laboratory of Structural Bioinformatics of Proteins, Institute of Biotechnology of the Czech Academy of Sciences, 14220 Prague, Czech Republic;  Orcidhttps://orcid.org/0000-0002-1969-9304
  • Author Contributions

    Conceptualization - P.N. and M.P.; M.P. performed all experiments and analyzed data. J.C. performed homology modeling. M.P. and P.N. wrote and edited the manuscript. All authors have approved the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Dr. Michael Volný and Carlos V. Melo for helpful discussions and editing of the manuscript. This work was mainly financially supported by the NPO-NEURO-EXCELLES (LX22NPO5107), Czech Science Foundation (22-27695S), the European Commission H2020 (EU_FT-ICR_MS grant agreement ID: 731077 and EPIC-XS - grant agreement ID: 823839). Additional institutional and facility support from the Academy of Sciences of the Czech Republic (RVO: 61388971), Grant Agency of Charles University (359521), the Ministry of Education of the Czech Republic (Structural mass spectrometry CF - LM2018127 CIISB), ELIXIR-CZ (LM2023055), and the European Regional Development Funds (CZ.1.05/1.1.00/02.0109 BIOCEV) are gratefully acknowledged. We also thank Julie Winterová for helpful statistical data analysis.

References

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This article references 65 other publications.

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    Chen, Jiawei; Cui, Weidong; Giblin, Daryl; Gross, Michael L.
    Journal of the American Society for Mass Spectrometry (2012), 23 (8), 1306-1318CODEN: JAMSEF; ISSN:1044-0305. (Springer)
    We report a new approach for the fast photochem. oxidn. of proteins (FPOP) whereby iodine species are used as the modifying reagent. We generate the radicals by photolysis of iodobenzoic acid at 248 nm; the putative iodine radical then rapidly modifies the target protein. This iodine-radical labeling is sensitive, tunable, and site-specific, modifying only histidine and tyrosine residues in contrast to OH radicals that modify 14 amino-acid side chains. We iodinated myoglobin (Mb) and apomyoglobin (aMb) in their native states and analyzed the outcome by both top-down and bottom-up proteomic strategies. Top-down sequencing selects a certain level (addn. of one I, two I's) of modification and dets. the major components produced in the modification reaction, whereas bottom-up reveals details for each modification site. Tyr146 is found to be modified for aMb but less so for Mb. His82, His93, and His97 are at least 10 times more modified for aMb than for Mb, in agreement with NMR studies. For carbonic anhydrase and its apo form, there are no significant differences of the modification extents, indicating their similarity in conformation and providing a control for this approach. For lispro insulin, insulin-EDTA, and insulin complexed with zinc, iodination yields are sensitive to differences in insulin oligomerization state. The iodine radical labeling is a promising addn. to protein footprinting methods, offering higher specificity and lower reactivity than ·OH and SO, two other radicals already employed in FPOP.
  15. 15
    Manzi, L.; Barrow, A. S.; Hopper, J. T. S.; Kaminska, R.; Kleanthous, C.; Robinson, C. V.; Moses, J. E.; Oldham, N. J. Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein. Angew. Chemie Int. Ed. 2017, 56 (47), 14873– 14877,  DOI: 10.1002/anie.201708254
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    Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein
    Manzi, Lucio; Barrow, Andrew S.; Hopper, Jonathan T. S.; Kaminska, Renata; Kleanthous, Colin; Robinson, Carol V.; Moses, John E.; Oldham, Neil J.
    Angewandte Chemie, International Edition (2017), 56 (47), 14873-14877CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Mapping the interaction sites between membrane-spanning proteins is a key challenge in structural biol. A carbene-footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilized in micelles. The diazirine-based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labeling of the membrane-spanning regions of the protein upon irradn. at 349 nm. Areas assocd. with protein-protein interactions between the trimer subunits remained unlabeled, thus revealing their location.
  16. 16
    Zhang, M. M.; Rempel, D. L.; Gross, M. L. A Fast Photochemical Oxidation of Proteins (FPOP) Platform for Free-Radical Reactions: The Carbonate Radical Anion with Peptides and Proteins. Free Radic. Biol. Med. 2019, 131, 126– 132,  DOI: 10.1016/j.freeradbiomed.2018.11.031
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    A Fast Photochemical Oxidation of Proteins (FPOP) platform for free-radical reactions: the carbonate radical anion with peptides and proteins
    Zhang, Mengru Mira; Rempel, Don L.; Gross, Michael L.
    Free Radical Biology & Medicine (2019), 131 (), 126-132CODEN: FRBMEH; ISSN:0891-5849. (Elsevier B.V.)
    Fast Photochem. Oxidn. of Protein (FPOP), based on a pulsed KrF laser (248 nm) for free-radical generation, is a biophys. method that utilizes hydroxyl radicals to footprint proteins in soln. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity detn. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast ("snapshot") footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradn. of small plugs of the targeted sample; iii) the use of methionine and catalase after radical oxidn. chem. to prevent post-oxidn. with residual oxidizing species; and iv) the utilization of mature mass spectrometry-based proteomic methods to afford detailed anal. In addn. to •OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biol. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO-•3. Other arom. amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliph. amino acids are inert, unlike with •OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.
  17. 17
    Cheng, M.; Zhang, B.; Cui, W.; Gross, M. L. Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting. Angew. Chemie - Int. Ed. 2017, 56 (45), 14007– 14010,  DOI: 10.1002/anie.201706697
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    17
    Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting
    Cheng, Ming; Zhang, Bojie; Cui, Weidong; Gross, Michael L.
    Angewandte Chemie, International Edition (2017), 56 (45), 14007-14010CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Described is a novel, laser-initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. *CF3 reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to .OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chem. and structural biol. and biotechnol. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.
  18. 18
    Fojtík, L.; Fiala, J.; Pompach, P.; Chmelík, J.; Matoušek, V.; Beier, P.; Kukačka, Z.; Novák, P. Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules. J. Am. Chem. Soc. 2021, 143 (49), 20670– 20679,  DOI: 10.1021/jacs.1c07771
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    Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules
    Fojtik, Lukas; Fiala, Jan; Pompach, Petr; Chmelik, Josef; Matousek, Vaclav; Beier, Petr; Kukacka, Zdenek; Novak, Petr
    Journal of the American Chemical Society (2021), 143 (49), 20670-20679CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure detn. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resoln. to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decompn. of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-Hb complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.
  19. 19
    Sharp, J. S.; Becker, J. M.; Hettich, R. L. Protein Surface Mapping by Chemical Oxidation: Structural Analysis by Mass Spectrometry. Anal. Biochem. 2003, 313 (2), 216– 225,  DOI: 10.1016/S0003-2697(02)00612-7
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    19
    Protein surface mapping by chemical oxidation: Structural analysis by mass spectrometry
    Sharp, Joshua S.; Becker, Jeffrey M.; Hettich, Robert L.
    Analytical Biochemistry (2003), 313 (2), 216-225CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
    The solvent-accessible surface area of proteins is important in biol. function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chem. technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidn. Under conditions of low protein oxidn. (zero to three oxygen atoms added per apomyoglobin mol.), we have pos. identified five oxidn. sites by liq. chromatog.-tandem mass spectrometry and high-resoln. Fourier transform mass spectrometry. Our results indicate that all oxidized amino acids, with the exception of methionine, have highly solvent-accessible side chains, but the rate of oxidn. may not be dictated solely by solvent accessibility and amino acid identity.
  20. 20
    Hambly, D. M.; Gross, M. L. Laser Flash Photolysis of Hydrogen Peroxide to Oxidize Protein Solvent-Accessible Residues on the Microsecond Timescale. J. Am. Soc. Mass Spectrom. 2005, 16 (12), 2057– 2063,  DOI: 10.1016/j.jasms.2005.09.008
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    Laser Flash Photolysis of Hydrogen Peroxide to Oxidize Protein Solvent-Accessible Residues on the Microsecond Timescale
    Hambly, David M.; Gross, Michael L.
    Journal of the American Society for Mass Spectrometry (2005), 16 (12), 2057-2063CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    Footprinting of proteins by hydroxyl radicals generated on the millisecond to minute timescales to probe protein surfaces suffers from the uncertainty that radical reactions cause the protein to unfold, exposing residues that are protected in the native protein. To circumvent this possibility, the authors developed a method using a 248 nm KrF excimer laser to cleave hydrogen peroxide at low concns. (15 mM, 0.04%), affording hydroxyl radicals that modify the protein in less than a microsecond. In the presence of a scavenger (20 mM glutamine), the radical lifetimes decrease to ∼1 μs, yet the reaction timescales are sufficient to provide significant oxidn. of the protein. These times are arguably faster than super-secondary protein structure can unfold as a result of the modification. The radical formation step takes place in a nanoliter flow cell so that only one laser pulse irradiates each bolus of sample. The oxidn. sites are located using std. anal. proteomics, requiring less than a nanomole of protein. The authors tested the method with apomyoglobin and obsd. modifications in accord with solvent accessibility data obtained from the crystal structure of holomyoglobin. Addnl., the results indicate that the F-helix is conformationally flexible in apomyoglobin, in accord with NMR results. The authors also find that the binding pocket is resistant to modifications, indicating that the protein pocket closes in the absence of the heme group - conclusions that cannot be drawn from current structural methods. When developed further, this method may enable the detn. of protein-ligand interfaces, affinity consts., folding pathways, and regions of conformational flexibility.
  21. 21
    Wang, L.; Chance, M. R. Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment. Mol. Cell. Proteomics 2017, 16 (5), 706– 716,  DOI: 10.1074/mcp.O116.064386
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    Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment
    Wang, Liwen; Chance, Mark R.
    Molecular & Cellular Proteomics (2017), 16 (5), 706-716CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
    Protein footprinting mediated by mass spectrometry has evolved over the last 30 years from proof of concept to commonplace biophysics tool, with unique capabilities for assessing structure and dynamics of purified proteins in physiol. states in soln. This review outlines the history and current capabilities of two major methods of protein footprinting: reversible hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (HRF), an irreversible covalent labeling approach. Technol. advances in both approaches now permit high-resoln. assessments of protein structure including secondary and tertiary structure stability mediated by backbone interactions (measured via HDX) and solvent accessibility of side chains (measured via HRF). Applications across many academic fields and in biotechnol. drug development are illustrated including: detection of protein interfaces, identification of ligand/drug binding sites, and monitoring dynamics of protein conformational changes along with future prospects for advancement of protein footprinting in structural biol. and biophysics research.
  22. 22
    Liu, X. R.; Zhang, M. M.; Gross, M. L. Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications. Chem. Rev. 2020, 120 (10), 4355– 4454,  DOI: 10.1021/acs.chemrev.9b00815
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    Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications
    Liu, Xiaoran Roger; Zhang, Mengru Mira; Gross, Michael L.
    Chemical Reviews (Washington, DC, United States) (2020), 120 (10), 4355-4454CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Proteins adopt different higher-order structures (HOS) to enable their unique biol. functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS detn. of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS anal., through which different covalent labeling approaches "mark" the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen-deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resoln., the authors present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biol. questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a ref. for investigators seeking a MS-based tool to address structural questions in protein science.
  23. 23
    Xu, G.; Chance, M. R. Hydroxyl Radical-Mediated Modification of Proteins as Probes for Structural Proteomics. Chem. Rev. 2007, 107 (8), 3514– 3543,  DOI: 10.1021/cr0682047
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    Hydroxyl Radical-Mediated Modification of Proteins as Probes for Structural Proteomics
    Xu, Guozhong; Chance, Mark R.
    Chemical Reviews (Washington, DC, United States) (2007), 107 (8), 3514-3543CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review including major sections on background and history of protein footprinting, generation of hydroxy radicals in soln., hydroxy radical mediated cleavage of the main chain and modification of side chains, and future prospects.
  24. 24
    Xu, G.; Chance, M. R. Radiolytic Modification of Acidic Amino Acid Residues in Peptides: Probes for Examining Protein–Protein Interactions. Anal. Chem. 2004, 76 (5), 1213– 1221,  DOI: 10.1021/ac035422g
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    24
    Radiolytic Modification of Acidic Amino Acid Residues in Peptides: Probes for Examining Protein-Protein Interactions
    Xu, Guozhong; Chance, Mark R.
    Analytical Chemistry (2004), 76 (5), 1213-1221CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Hydroxyl radical-mediated footprinting coupled with mass spectroscopic anal. is a new technique for mapping protein surfaces, identifying structural changes modulated by protein-ligand binding, and mapping protein-ligand interfaces in soln. In this study, the authors examine the radiolytic oxidn. of aspartic and glutamic acid residues to probe their potential use as structural probes in footprinting expts. Model peptides contg. Asp or Glu were irradiated using white light from a synchrotron x-ray source or a cesium-137 γ-ray source. The radiolysis products were characterized by electrospray mass spectrometry including tandem mass spectrometry. Both Asp and Glu are susceptible to radiolytic oxidization by γ-rays or synchrotron x-rays. Radiolysis results primarily in the oxidative decarboxylation of the side chain carboxyl group and formation of an aldehyde group at the carbon next to the original carboxyl group, giving rise to a characteristic product with a -30 Da mass change. A similar oxidative decarboxylation also takes place for amino acids with C-terminal carboxyl groups. The methylene groups in the Asp and Glu side chains also undergo oxygen addn. forming ketone or alc. groups with mass changes of +14 and +16 Da, resp. Characterizing the oxidn. reactions of these two acidic residues extends the no. of useful side chain probes for hydroxyl radical-mediated protein footprinting from 10 (Cys, Met, Trp, Tyr, Phe, Arg, Leu, Pro, His, Lys) to 12 amino acid residues, thus enhancing the ability to map protein surface structure, and in combination with previously identified basic amino acid probes, this method can be used to examine mol. details of protein-protein interactions that are driven by electrostatics.
  25. 25
    Xu, G.; Takamoto, K.; Chance, M. R. Radiolytic Modification of Basic Amino Acid Residues in Peptides: Probes for Examining Protein–Protein Interactions. Anal. Chem. 2003, 75 (24), 6995– 7007,  DOI: 10.1021/ac035104h
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    Radiolytic modification of basic amino acid residues in peptides: Probes for examining protein-protein interactions
    Xu, Guozhong; Takamoto, Keiji; Chance, Mark R.
    Analytical Chemistry (2003), 75 (24), 6995-7007CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Protein footprinting utilizing hydroxyl radicals coupled with mass spectrometry has become a powerful technique for mapping the solvent accessible surface of proteins and examg. protein-protein interactions in soln. Hydroxyl radicals generated by radiolysis or chem. methods efficiently react with many amino acid residue side chains, including the arom. and sulfur-contg. residues along with proline and leucine, generating stable oxidn. products that are valuable probes for examg. protein structure. In this study, we examine the radiolytic oxidn. chem. of histidine, lysine, and arginine for comparison with their metal-catalyzed oxidn. products. Model peptides contg. arginine, histidine, and lysine were irradiated using white light from a synchrotron x-ray source or a cesium-137 γ-ray source. The rates of oxidn. and the radiolysis products were primarily characterized by electrospray mass spectrometry including tandem mass spectrometry. Arginine is very sensitive to radiolytic oxidn., giving rise to a characteristic product with a 43 Da mass redn. as a result of the loss of guanidino group and conversion to γ-glutamyl semialdehyde, consistent with previous metal-catalyzed oxidn. studies. Histidine was oxidized to generate a mixt. of products with characteristic mass changes primarily involving rupture of and addn. to the imidazole ring. Lysine was converted to hydroxylysine or carbonylysine by radiolysis. The development of methods to probe these residues due to their high frequency of occurrence, their typical presence on the protein surface, and their frequent participation in protein-protein interactions considerably extends the utility of protein footprinting.
  26. 26
    Xu, G.; Chance, M. R. Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting. Anal. Chem. 2005, 77 (8), 2437– 2449,  DOI: 10.1021/ac0484629
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    Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting
    Xu, Guozhong; Chance, Mark R.
    Analytical Chemistry (2005), 77 (8), 2437-2449CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Protein footprinting based on hydroxyl radical-mediated modification and quant. mass spectroscopic anal. is a proven technique for examg. protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural anal. depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-contg. amino acids are oxidized readily and the mechanisms of oxidn. are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here the authors have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides contg. cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidn. using γ-rays or synchrotron x-rays and have compared these results to those expected from oxidn. mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are obsd. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidn. is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the γ-carbon. Due to the high reactivity of sulfur-contg. amino acids, the extent of oxidn. is easily influenced by secondary oxidn. events or the presence of redox reagents used in std. proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine ∼ tryptophan > cystine is obsd.
  27. 27
    Charvátová, O.; Foley, B. L.; Bern, M. W.; Sharp, J. S.; Orlando, R.; Woods, R. J. Quantifying Protein Interface Footprinting by Hydroxyl Radical Oxidation and Molecular Dynamics Simulation: Application to Galectin-1. J. Am. Soc. Mass Spectrom. 2008, 19 (11), 1692– 1705,  DOI: 10.1016/j.jasms.2008.07.013
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    Quantifying Protein Interface Footprinting by Hydroxyl Radical Oxidation and Molecular Dynamics Simulation: Application to Galectin-1
    Charvatova, Olga; Foley, B. Lachele; Bern, Marshall W.; Sharp, Joshua S.; Orlando, Ron; Woods, Robert J.
    Journal of the American Society for Mass Spectrometry (2008), 19 (11), 1692-1705CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    Biomol. surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to det. high-resoln. three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resoln. compared with traditional chem. derivatization. Here the authors present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissocn. of hydrogen peroxide. The authors applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient anal. of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large no. of modifications arising from side-chain oxidn. Quantification of the level of oxidn. has been achieved by employing spectral intensities for all of the obsd. oxidn. states on a per-residue basis. The level of accuracy achievable from spectral intensities was detd. by examn. of mixts. of synthetic peptides related to those present after oxidn. and tryptic digestion of galectin-1. A direct relation between side-chain solvent accessibility and level of oxidn. emerged, which enabled the prediction of the level of oxidn. given the 3D structure of the protein. The precision of this relation was enhanced through the use of av. solvent accessibilities computed from 10 ns mol. dynamics simulations of the protein.
  28. 28
    Pan, Y.; Stocks, B. B.; Brown, L.; Konermann, L. Structural Characterization of an Integral Membrane Protein in Its Natural Lipid Environment by Oxidative Methionine Labeling and Mass Spectrometry. Anal. Chem. 2009, 81 (1), 28– 35,  DOI: 10.1021/ac8020449
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    Structural Characterization of an Integral Membrane Protein in Its Natural Lipid Environment by Oxidative Methionine Labeling and Mass Spectrometry
    Pan, Yan; Stocks, Bradley B.; Brown, Leonid; Konermann, Lars
    Analytical Chemistry (Washington, DC, United States) (2009), 81 (1), 28-35CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Membrane proteins represent formidable challenges for many anal. techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a nonnatural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-contg. purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dil. aq. H2O2. The expts. rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following ·OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-sol. proteins, which generally undergo modifications at many different types of residues. In those earlier expts. the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidn. pattern obsd. here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidn. The authors' results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, esp. when considering that many of these species are methionine-rich. The introduction of addnl. Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodol. described here.
  29. 29
    Watkinson, T. G.; Calabrese, A. N.; Ault, J. R.; Radford, S. E.; Ashcroft, A. E. FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins. J. Am. Soc. Mass Spectrom. 2017, 28 (1), 50– 55,  DOI: 10.1007/s13361-016-1421-1
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    FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins
    Watkinson, Thomas G.; Calabrese, Antonio N.; Ault, James R.; Radford, Sheena E.; Ashcroft, Alison E.
    Journal of the American Society for Mass Spectrometry (2017), 28 (1), 50-55CODEN: JAMSEF; ISSN:1044-0305. (Springer)
    Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in soln., and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochem. oxidn. of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quant. mass spectrometry analyses, we show that fast photochem. oxidn. reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of addnl. intermol. contacts.
  30. 30
    Lu, Y.; Zhang, H.; Niedzwiedzki, D. M.; Jiang, J.; Blankenship, R. E.; Gross, M. L. Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc. Anal. Chem. 2016, 88 (17), 8827– 8834,  DOI: 10.1021/acs.analchem.6b01945
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    Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc
    Lu, Yue; Zhang, Hao; Niedzwiedzki, Dariusz M.; Jiang, Jing; Blankenship, Robert E.; Gross, Michael L.
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (17), 8827-8834CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Although membrane proteins are crucial participants in photosynthesis and other biol. processes, many lack high-resoln. structures. Prior to achieving a high-resoln. structure, the authors are studying whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. The authors' platform probes topol. and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochem. oxidn. of proteins (FPOP), and lipid Nanodiscs, which more similar to the native membrane environment than are the widely used detergent micelles. The authors describe here results that show a protein's outer membrane regions are more heavily footprinted by OH radicals whereas the regions spanning the lipid bilayer remain inert to the labeling. Nanodiscs generally exhibit more protection of membrane proteins compared to detergent micelles and less shielding to those protein residues that exist outside the membrane. The combination of immobilizing the protein in Nanodiscs and footprinting with the FPOP approach is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level, and to illuminate intrinsic membrane protein topol.
  31. 31
    Gupta, S.; Bavro, V. N.; D’Mello, R.; Tucker, S. J.; Vénien-Bryan, C.; Chance, M. R. Conformational Changes during the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry. Structure 2010, 18 (7), 839– 846,  DOI: 10.1016/j.str.2010.04.012
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    Conformational Changes During the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry
    Gupta, Sayan; Bavro, Vassiliy N.; D'Mello, Rhijuta; Tucker, Stephen J.; Venien-Bryan, Catherine; Chance, Mark R.
    Structure (Cambridge, MA, United States) (2010), 18 (7), 839-846CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
    Summary: Potassium channels are dynamic proteins that undergo large conformational changes to regulate the flow of K+ ions across the cell membrane. Understanding the gating mechanism of these channels therefore requires methods for probing channel structure in both their open and closed conformations. Radiolytic footprinting is used to study the gating mechanism of the inwardly-rectifying potassium channel KirBac3.1. The purified protein stabilized in either open or closed conformations was exposed to focused synchrotron X-ray beams on millisecond timescales to modify solvent accessible amino acid side chains. These modifications were identified and quantified using high-resoln. mass spectrometry. The differences obsd. between the closed and open states were then used to reveal local conformational changes that occur during channel gating. The results provide support for a proposed gating mechanism of the Kir channel and demonstrate a method of probing the dynamic gating mechanism of other integral membrane proteins and ion channels.
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    Loginov, D. S.; Fiala, J.; Brechlin, P.; Kruppa, G.; Novak, P. Hydroxyl Radical Footprinting Analysis of a Human Haptoglobin-Hemoglobin Complex. Biochim. Biophys. Acta - Proteins Proteomics 2022, 1870 (2), 140735  DOI: 10.1016/j.bbapap.2021.140735
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    Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex
    Loginov, Dmitry S.; Fiala, Jan; Brechlin, Peter; Kruppa, Gary; Novak, Petr
    Biochimica et Biophysica Acta, Proteins and Proteomics (2022), 1870 (2), 140735CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
    Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochem. oxidn. of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile detn. of the site of modification with single residue resoln. In the present study, FPOP anal. was applied to study the Hb (Hb) - haptoglobin (Hp) complex allowing identification of resp. regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, resp., including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed detn. of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.
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    Cornwell, O.; Bond, N. J.; Radford, S. E.; Ashcroft, A. E. Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS. Anal. Chem. 2019, 91 (23), 15163– 15170,  DOI: 10.1021/acs.analchem.9b03958
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    Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS
    Cornwell, Owen; Bond, Nicholas J.; Radford, Sheena E.; Ashcroft, Alison E.
    Analytical Chemistry (Washington, DC, United States) (2019), 91 (23), 15163-15170CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochem. Oxidn. of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labeling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal down-stream anal. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity detg. regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-assocn. at high concns. and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labeling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labeling patterns at the interfaces of the CL-CH1 and CH1-CH2 domains, with the nonaggregating variant undergoing up to four times more labeling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL - CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidized peptides. Some oxidized amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an anal. as an aid to MS/MS in assigning oxidn. sites.
  34. 34
    Cornwell, O.; Radford, S. E.; Ashcroft, A. E.; Ault, J. R. Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: A Structural Characterisation of Wild-Type and ΔN6 B2-Microglobulin. J. Am. Soc. Mass Spectrom. 2018, 29 (12), 2413– 2426,  DOI: 10.1007/s13361-018-2067-y
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    Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and ΔN6 β2-Microglobulin
    Cornwell, Owen; Radford, Sheena E.; Ashcroft, Alison E.; Ault, James R.
    Journal of the American Society for Mass Spectrometry (2018), 29 (12), 2413-2426CODEN: JAMSEF; ISSN:1044-0305. (Springer)
    Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochem. oxidn. of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β2-microglobulin (β2m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimized LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures. [Figure not available: see fulltext.].
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    Yassaghi, G.; Kukačka, Z.; Fiala, J.; Kavan, D.; Halada, P.; Volný, M.; Novák, P. Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation. Anal. Chem. 2022, 94 (28), 9993– 10002,  DOI: 10.1021/acs.analchem.1c05476
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    Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation
    Yassaghi, Ghazaleh; Kukacka, Zdenek; Fiala, Jan; Kavan, Daniel; Halada, Petr; Volny, Michael; Novak, Petr
    Analytical Chemistry (Washington, DC, United States) (2022), 94 (28), 9993-10002CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Fast photochem. oxidn. of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chem. reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established std. method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixt. of peptides from various subpopulations of proteins with varying degrees of photochem. oxidn. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down expt. is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissocn., electron-transfer dissocn., and electron-capture dissocn. combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.
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    Tomášková, N.; Novák, P.; Kožár, T.; Petrenčáková, M.; Jancura, D.; Yassaghi, G.; Man, P.; Sedlák, E. Early Modification of Cytochrome c by Hydrogen Peroxide Triggers Its Fast Degradation. Int. J. Biol. Macromol. 2021, 174, 413– 423,  DOI: 10.1016/j.ijbiomac.2021.01.189
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    Early modification of cytochrome c by hydrogen peroxide triggers its fast degradation
    Tomaskova, Natasa; Novak, Petr; Kozar, Tibor; Petrencakova, Martina; Jancura, Daniel; Yassaghi, Ghazaleh; Man, Petr; Sedlak, Erik
    International Journal of Biological Macromolecules (2021), 174 (), 413-423CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
    Cytochrome c (cyt c), in addn. to its function as an electron shuttle in respiratory chain, is able to perform as a pseudo-peroxidase with a crit. role during apoptosis. Incubation of cyt c with an excess of hydrogen peroxide leads to a suicide inactivation of the protein, which is accompanied by heme destruction and covalent modification of numerous amino acid residues. Although steady-state reactions of cyt c with an excess of hydrogen peroxide represent non-physiol. conditions, they might be used for anal. of the first-modified amino acid in in vivo. Here, we obsd. oxidn. of tyrosine residues 67 and 74 and heme as the first modifications found upon incubation with hydrogen peroxide. The positions of the oxidized tyrosines suggest a possible migration pathway of hydrogen peroxide-induced radicals from the site of heme localization to the protein surface. Anal. of a size of folded fraction of cyt c upon limited incubation with hydrogen peroxide indicates that the early oxidn. of amino acids triggers an accelerated destruction of cyt c. Position of channels from mol. dynamics simulation structures of cyt c points to a location of amino acid residues exposed to reactive oxidants that are thus more prone to covalent modification.
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    Donnelly, D. P.; Rawlins, C. M.; DeHart, C. J.; Fornelli, L.; Schachner, L. F.; Lin, Z.; Lippens, J. L.; Aluri, K. C.; Sarin, R.; Chen, B.; Lantz, C.; Jung, W.; Johnson, K. R.; Koller, A.; Wolff, J. J.; Campuzano, I. D. G.; Auclair, J. R.; Ivanov, A. R.; Whitelegge, J. P.; Paša-Tolić, L.; Chamot-Rooke, J.; Danis, P. O.; Smith, L. M.; Tsybin, Y. O.; Loo, J. A.; Ge, Y.; Kelleher, N. L.; Agar, J. N. Best Practices and Benchmarks for Intact Protein Analysis for Top-down Mass Spectrometry. Nat. Methods 2019, 16 (7), 587– 594,  DOI: 10.1038/s41592-019-0457-0
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    Best practices and benchmarks for intact protein analysis for top-down mass spectrometry
    Donnelly, Daniel P.; Rawlins, Catherine M.; DeHart, Caroline J.; Fornelli, Luca; Schachner, Luis F.; Lin, Ziqing; Lippens, Jennifer L.; Aluri, Krishna C.; Sarin, Richa; Chen, Bifan; Lantz, Carter; Jung, Wonhyeuk; Johnson, Kendall R.; Koller, Antonius; Wolff, Jeremy J.; Campuzano, Iain D. G.; Auclair, Jared R.; Ivanov, Alexander R.; Whitelegge, Julian P.; Pasa-Tolic, Ljiljana; Chamot-Rooke, Julia; Danis, Paul O.; Smith, Lloyd M.; Tsybin, Yury O.; Loo, Joseph A.; Ge, Ying; Kelleher, Neil L.; Agar, Jeffrey N.
    Nature Methods (2019), 16 (7), 587-594CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic mol. mass. Top-down mass spectrometry enables the anal. of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass anal. of intact proteins (antibodies, membrane proteins and others) from mixts. of varying complexity. We also present cross-platform anal. benchmarks using a protein std. sample, to allow users to gauge their proficiency.
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    Petrenčáková, M.; Filandr, F.; Hovan, A.; Yassaghi, G.; Man, P.; Kožár, T.; Schwer, M. S.; Jancura, D.; Plückthun, A.; Novák, P.; Miškovský, P.; Bánó, G.; Sedlák, E. Photoinduced Damage of AsLOV2 Domain Is Accompanied by Increased Singlet Oxygen Production Due to Flavin Dissociation. Sci. Rep. 2020, 10 (1), 1– 15,  DOI: 10.1038/s41598-020-60861-2
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    Kellersberger, K. A.; Yu, E.; Kruppa, G. H.; Young, M. M.; Fabris, D. Top-Down Characterization of Nucleic Acids Modified by Structural Probes Using High-Resolution Tandem Mass Spectrometry and Automated Data Interpretation. Anal. Chem. 2004, 76 (9), 2438– 2445,  DOI: 10.1021/ac0355045
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    Top-Down Characterization of Nucleic Acids Modified by Structural Probes Using High-Resolution Tandem Mass Spectrometry and Automated Data Interpretation
    Kellersberger, Katherine A.; Yu, Eizadora; Kruppa, Gary H.; Young, Malin M.; Fabris, Daniele
    Analytical Chemistry (2004), 76 (9), 2438-2445CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A top-down approach based on sustained off-resonance irradn. collision-induced dissocn. (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as di-Me sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and β-ethoxy-α-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chem. to produce strand cleavage at the modified nucleotides, and anal. by PAGE to det. the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the anal. of individual target adducts present in complex reaction mixts. with no need for sepn. techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MSn expts. to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated redn. and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data redn. module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calcs. the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data redn. program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on crosslinking strategies and top-down ESI-FTMS.
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    Valkenborg, D.; Mertens, I.; Lemière, F.; Witters, E.; Burzykowski, T. The Isotopic Distribution Conundrum. Mass Spectrometry Reviews; John Wiley & Sons, Ltd, January 1, 2012; pp 96– 109.  DOI: 10.1002/mas.20339 .
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    Compton, P. D.; Zamdborg, L.; Thomas, P. M.; Kelleher, N. L. On the Scalability and Requirements of Whole Protein Mass Spectrometry. Anal. Chem. 2011, 83 (17), 6868– 6874,  DOI: 10.1021/ac2010795
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    On the Scalability and Requirements of Whole Protein Mass Spectrometry
    Compton, Philip D.; Zamdborg, Leonid; Thomas, Paul M.; Kelleher, Neil L.
    Analytical Chemistry (Washington, DC, United States) (2011), 83 (17), 6868-6874CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Top-down proteomics has improved over the past decade despite the significant challenges presented by the anal. of large protein ions. Here, the detection of these high mass species by electrospray-based mass spectrometry (MS) is examd. from a theor. perspective to understand the mass-dependent increases in the no. of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quant. model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of 13C and 15N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, the authors find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20-fold.
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    Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S.; Guan, S.; Logan, T. M. Protein Molecular Mass to 1 Da by 13 C, 15 N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119 (2), 433– 434,  DOI: 10.1021/ja9630046
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    Protein Molecular Weight to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry
    Marshall, Alan G.; Senko, Michael W.; Li, Weiqun; Li, Ming; Dillon, Stephanie; Guan, Shenheng; Logan, Timothy M.
    Journal of the American Chemical Society (1997), 119 (2), 433-434CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Although electrosprayed protein gas-phase ion masses can be detd. to ppm accuracy, the broad natural-abundance isotopic distribution can lead to significant error (≥1 Da) in detn. of the protein monoisotopic mol. wt. Here, we show that a protein doubly-depleted in 13C and 15N exhibits a substantially narrowed isotopic distribution with greatly increased population of the monoisotopic species (all carbons are 12C, all nitrogens are 14N, all oxygens are 16O, etc.). The narrower isotopic distribution will facilitate MS/MS and adduct identification, and promises to extend substantially the upper limit for protein mass anal.
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    Bou-Assaf, G. M.; Chamoun, J. E.; Emmett, M. R.; Fajer, P. G.; Marshall, A. G. Advantages of Isotopic Depletion of Proteins for Hydrogen/Deuterium Exchange Experiments Monitored by Mass Spectrometry. Anal. Chem. 2010, 82 (8), 3293– 3299,  DOI: 10.1021/ac100079z
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    Advantages of Isotopic Depletion of Proteins for Hydrogen/Deuterium Exchange Experiments Monitored by Mass Spectrometry
    Bou-Assaf, George M.; Chamoun, Jean E.; Emmett, Mark R.; Fajer, Piotr G.; Marshall, Alan G.
    Analytical Chemistry (Washington, DC, United States) (2010), 82 (8), 3293-3299CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Soln.-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein-protein interactions and conformational changes in biol. systems, esp. when traditional methods such as x-ray crystallog. or NMR are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein mol. wt.(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addn., different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species (12Cc1Hh14Nn16Oo32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data redn.
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    Charlebois, J. P.; Patrie, S. M.; Kelleher, N. L. Electron Capture Dissociation and 13C,15N Depletion for Deuterium Localization in Intact Proteins after Solution-Phase Exchange. Anal. Chem. 2003, 75 (13), 3263– 3266,  DOI: 10.1021/ac020690k
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    Electron Capture Dissociation and 13C, 15N Depletion for Deuterium Localization in Intact Proteins after Solution-Phase Exchange
    Charlebois, Jay P.; Patrie, Steven M.; Kelleher, Neil L.
    Analytical Chemistry (2003), 75 (13), 3263-3266CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    For localization of deuterium atoms after soln.-phase exchange with D2O, intact proteins are often digested prior to anal. by mass spectrometry (MS) and tandem MS (MS/MS). Amelioration of limitations assocd. with this approach (e.g., <70% sequence coverage and some D atom scrambling during MS/MS) were sought using intact proteins and two newer methods applied to tracking H/D exchange dynamics for the first time. Using 2-4-fold signal enhancements through depletion of 13C and 15N isotopes and implementing the new MS/MS technique of electron capture dissocn. (ECD) yielded an increased no. of c and z• ions obsd. (43 vs. 25) for recombinant yeast ubiquitin (9.3 kDa). Initial detn. of D atom content in consecutive c ion series (c4 - c7, c28, c31, c32, and c33) was demonstrated. The improved ion signal and expt. speed combined with narrower isotopic distributions markedly increases the degree of localization and feasibility of ECD-based MS/MS after soln.-phase H/D exchange.
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    Zubarev, R. A.; Demirev, P. A. Isotope Depletion of Large Biomolecules: Implications for Molecular Mass Measurements. J. Am. Soc. Mass Spectrom. 1998, 9 (2), 149– 156,  DOI: 10.1016/S1044-0305(97)00232-8
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    Isotope depletion of large biomolecules: implications for molecular mass measurements
    Zubarev, Roman A.; Demirev, Plamen A.
    Journal of the American Society for Mass Spectrometry (1998), 9 (2), 149-156CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
    Isotope depletion (or enrichment) of large biomols. is a procedure already used in high resoln. Fourier transform ion cyclotron resonance mass spectrometry for improving the reliability and accuracy of biomol. mass characterization. In this work, effects of isotope depletion on a no. of mass spectrometric parameters are systematically studied. Implementation of the isotope depletion techniques in conjunction with lower resoln. mass analyzers is discussed as well. The authors investigate theor. the position of the centroid of the isotopic mass distributions (centroid mass) and the shift between the monoisotopic and the centroid masses of biopolymers as a function of the isotope abundance (e.g., 12C:13C ratio). The behavior of other additive mass parameters, like the ratio between the monoisotopic and the first isotopic peak, is also discussed. We address by computer simulations the effects of different instrumental parameters like mass resoln. and ion statistics as a function of isotope abundances and from there the achievable mass accuracy for high-mass biopolymers. We assess some of the practical issues of the isotope depletion technique, viz., to what degree and with what accuracy the depletion procedure should be performed for achieving the desired mass accuracy.
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    Gallagher, K. J.; Palasser, M.; Hughes, S.; Mackay, C. L.; Kilgour, D. P. A.; Clarke, D. J. Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins. J. Am. Soc. Mass Spectrom. 2020, 31 (3), 700– 710,  DOI: 10.1021/jasms.9b00119
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    Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins
    Gallagher, Kelly J.; Palasser, Michael; Hughes, Sam; Mackay, C. Logan; Kilgour, David P. A.; Clarke, David J.
    Journal of the American Society for Mass Spectrometry (2020), 31 (3), 700-710CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
    Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biol. MS expts., the practicality of applying top-down methodologies is still limited at higher mol. mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing no. of isotopic peaks as mol. mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago and has been demonstrated for several small proteins. Here the authors extend this approach, introducing a new highly efficient method for the prodn. of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down anal. of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal obsd. up to 50 kDa. In top-down fragmentation expts., the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, the authors demonstrate greatly increased sequence coverage in time-limited top-down expts.-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.
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    Popovic, Z.; Anderson, L. C.; Zhang, X.; Butcher, D. S.; Blakney, G. T.; Zubarev, R. A.; Marshall, A. G. Analysis of Isotopically Depleted Proteins Derived from Escherichia Coli and Caenorhabditis Elegans Cell Lines by Liquid Chromatography 21 T Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2023, 34, 137– 144,  DOI: 10.1021/jasms.2c00242
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    Analysis of Isotopically Depleted Proteins Derived from Escherichia coli and Caenorhabditis elegans Cell Lines by Liquid Chromatography 21 T Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry
    Popovic, Zeljka; Anderson, Lissa C.; Zhang, Xuepei; Butcher, David S.; Blakney, Greg T.; Zubarev, Roman A.; Marshall, Alan G.
    Journal of the American Society for Mass Spectrometry (2023), 34 (2), 137-144CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
    Protein mass measurement by mass spectrometry is complicated by wide isotopic distributions that result from incorporation of heavy isotopes of C, H, N, O, and S, thereby limiting signal-to-noise ratio (SNR) and accurate intact mass detn., particularly for larger proteins []. Observation of the monoisotopic mass-to-charge ratio (m/z) is the simplest and most accurate way to det. intact protein mass, but as mass increases, the relative abundance of the monoisotopic peak becomes so low that it is often undetectable. Here, we used an isotopically depleted growth medium to culture bacterial cells (Escherichia coli), resulting in isotopically depleted proteins. Isotopically depleted proteins show increased sequence coverage, mass measurement accuracy, and increased S/N of the monoisotopic peak by Fourier transform ion cyclotron resonance mass spectrometry anal. We then grew Caenorhabditis elegans cells in a medium contg. living isotopically depleted E. coli cells, thereby producing the first isotopically depleted eukaryotic proteins. This is the first time isotopic depletion has been implemented for four isotopes (1H, 12C, 14N, and 16O), resulting in the highest degree of depletion ever used for protein anal. and further improving MS anal.
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    Loginov, D. S.; Fiala, J.; Chmelik, J.; Brechlin, P.; Kruppa, G.; Novak, P. Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on TimsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis. ACS Omega 2021, 6 (15), 10352– 10361,  DOI: 10.1021/acsomega.1c00732
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    Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis
    Loginov, Dmitry S.; Fiala, Jan; Chmelik, Josef; Brechlin, Peter; Kruppa, Gary; Novak, Petr
    ACS Omega (2021), 6 (15), 10352-10361CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    Fast photochem. oxidn. of proteins (FPOP) is a recently developed technique for studying protein folding, conformations, interactions, etc. In this method, hydroxyl radicals, usually generated by KrF laser photolysis of H2O2, are used for irreversible labeling of solvent-exposed side chains of amino acids. Mapping of the oxidized residues to the protein's structure requires pinpointing of modifications using a bottom-up proteomic approach. In this work, a quadrupole time-of-flight (QTOF) mass spectrometer coupled with trapped ion mobility spectrometry (timsTOF Pro) was used for identification of oxidative modifications in a model protein. Multiple modifications on the same residues, including six modifications of histidine, were successfully resolved. Moreover, parallel accumulation-serial fragmentation (PASEF) technol. allows successful sequencing of even minor populations of modified peptides. The data obtained indicate a clear improvement of the quality of the FPOP anal. from the viewpoint of the no. of identified peptides bearing oxidative modifications and their precise localization. Data are available via ProteomeXchange with identifier PXD020509.
  49. 49
    Li, K. S.; Shi, L.; Gross, M. L. Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization. Acc. Chem. Res. 2018, 51 (3), 736– 744,  DOI: 10.1021/acs.accounts.7b00593
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    49
    Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization
    Li, Ke Sherry; Shi, Liuqing; Gross, Michael L.
    Accounts of Chemical Research (2018), 51 (3), 736-744CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Assessment of protein structure and interaction is crucial for understanding protein structure/function relations. Compared to high-resoln. structural tools, including x-ray crystallog., NMR, and cryo-EM, and traditional low-resoln. methods, such as CD, UV-visible, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resoln. (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure. The methodol. relies on "painting" the reactive and solvent-exposed amino acid residues with chem. tags and using the pattern of modifications as footprints from anal. by bottom-up MS-based proteomics to deduce protein higher order structures. The outcome can refer to proteins in soln. or even in cells and is complementary to those of x-ray crystallog. and NMR. It is particularly useful in mapping protein-ligand interfaces and conformational changes resulting from ligand binding, mutation, and aggregation.Fast photochem. oxidn. of proteins (FPOP), in its original conception, is a type of hydroxyl-radical-based protein footprinting that uses a pulsed KrF laser (248 nm) to trigger hydrolysis of hydrogen peroxide to produce soln. hydroxyl radicals, which subsequently modify the protein in situ. The platform is expanding to adopt other reactive species including carbenes. The reactivity of the probe depends on the intrinsic reactivity of the radical with the residue side chain and the solvent accessibility of the residue as a function of the tertiary/quaternary structures. By introducing an appropriate scavenger to compete with hydroxyl radical self-quenching, the lifetime of the primary radicals is remarkably shortened to approx. microsecond. Thus, the sampling time scale of FPOP is much faster than hydrogen-deuterium exchange and other covalent labeling methods relying on nonradical reactions.The short footprinting time scale of FPOP offers two major advantages for protein structure elucidation: (1) it allows the protein to be interrogated in its native or near-native state with min. structural perturbation; (2) it exhibits high sensitivity toward alterations in protein higher order structures because its sampling time is short with respect to protein conformational changes and dynamic motion. In addn., the covalent and irreversible oxidn. by the hydroxyl radical provides more flexibility in the downstream proteomics workflow and MS anal., permitting high spatial resoln. with residue-specific information.Since its invention in 2005 by Hambly and Gross, FPOP has developed from proof-of-concept to a valuable biophys. tool for interrogating protein structure. In this Account, the authors summarize the principles and exptl. design of FPOP that enable its fast labeling and describe the current and unique capabilities of the technique in protein higher order structure elucidation. Application examples include characterization of amyloid β self-assembly, protein-ligand interactions with a special emphasis on epitope mapping for protein therapeutics (e.g., antibody, Fab, and adnectin), protein folding detailed to residue-specific folding kinetics, and protein flexibility/dynamics. Addnl., the utility of FPOP-based oxidative footprinting should grow with the authors' continuing developments of novel reagents (e.g., sulfate radical anion, carbene diradical, and trifluoromethyl radical). These reactive reagents are compatible with the current FPOP platform and offer different reactivity and selectivity toward various types of amino acid residues, providing complementary insights into protein higher order structures for sol. proteins and ultimately for membrane-bound proteins.
  50. 50
    Perez-Riverol, Y.; Bai, J.; Bandla, C.; García-Seisdedos, D.; Hewapathirana, S.; Kamatchinathan, S.; Kundu, D. J.; Prakash, A.; Frericks-Zipper, A.; Eisenacher, M.; Walzer, M.; Wang, S.; Brazma, A.; Vizcaíno, J. A. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50 (D1), D543– D552,  DOI: 10.1093/nar/gkab1038
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    PRIDE database resources in 2022 hub for mass spectrometry-based proteomics evidences
    Perez-Riverol, Yasset; Bai, Jingwen; Bandla, Chakradhar; Garcia-Seisdedos, David; Hewapathirana, Suresh; Kamatchinathan, Selvakumar; Kundu, Deepti J.; Prakash, Ananth; Frericks-Zipper, Anika; Eisenacher, Martin; Walzer, Mathias; Wang, Shengbo; Brazma, Alvis; Vizcaino, Juan Antonio
    Nucleic Acids Research (2022), 50 (D1), D543-D552CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    The PRoteomics IDEntifications (PRIDE) database is the world's largest data repository of mass spectrometry-based proteomics data. PRIDE is one of the founding members of the global ProteomeXchange (PX) consortium and an ELIXIR core data resource. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2019. The no. of submitted datasets to PRIDE Archive (the archival component of PRIDE) has reached on av. around 500 datasets per mo during 2021. In addn. to continuous improvements in PRIDE Archive data pipelines and infrastructure, the PRIDE Spectra Archive has been developed to provide direct access to the submitted mass spectra using Universal Spectrum Identifiers. As a key point, the file format MAGE-TAB for proteomics has been developed to enable the improvement of sample metadata annotation. Addnl., the resource PRIDE Peptidome provides access to aggregated peptide/protein evidences across PRIDE Archive. Furthermore, we will describe how PRIDE has increased its efforts to reuse and disseminate high-quality proteomics data into other added-value resources such as UniProt, Ensembl and Expression Atlas.
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    Boura, E.; Rezabkova, L.; Brynda, J.; Obsilova, V.; Obsil, T. Structure of the Human FOXO4-DBD–DNA Complex at 1.9 Å Resolution Reveals New Details of FOXO Binding to the DNA. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66 (12), 1351– 1357,  DOI: 10.1107/S0907444910042228
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    Structure of the human FOXO4-DBD-DNA complex at 1.9 Å resolution reveals new details of FOXO binding to the DNA
    Boura, Evzen; Rezabkova, Lenka; Brynda, Jiri; Obsilova, Veronika; Obsil, Tomas
    Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (12), 1351-1357CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    FOXO4 is a member of the FOXO subgroup of forkhead transcription factors that constitute key components of a conserved signaling pathway that connects growth and stress signals to transcriptional control. Here, the 1.9 Å resoln. crystal structure of the DNA-binding domain of human FOXO4 (FOXO4-DBD) bound to a 13 bp DNA duplex contg. a FOXO consensus binding sequence is reported. The structure shows a similar recognition of the core sequence as has been shown for two other FOXO proteins. Helix H3 is docked into the major groove and provides all of the base-specific contacts, while the N-terminus and wing W1 make addnl. contacts with the phosphate groups of DNA. In contrast to other FOXO-DBD-DNA structures, the loop between helixes H2 and H3 has a different conformation and participates in DNA binding. In addn., the structure of the FOXO4-DBD-DNA complex suggests that both direct water-DNA base contacts and the unique water-network interactions contribute to FOXO-DBD binding to the DNA in a sequence-specific manner.
  52. 52
    Flores, S. C.; Altman, R. B. Turning Limited Experimental Information into 3D Models of RNA. RNA 2010, 16 (9), 1769– 1778,  DOI: 10.1261/rna.2112110
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    Turning limited experimental information into 3D models of RNA
    Flores, Samuel Coulbourn; Altman, Russ B.
    RNA (2010), 16 (9), 1769-1778CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    Our understanding of RNA functions in the cell is evolving rapidly. As for proteins, the detailed three-dimensional (3D) structure of RNA is often key to understanding its function. Although crystallog. and NMR can det. the at. coordinates of some RNA structures, many 3D structures present tech. challenges that make these methods difficult to apply. The great flexibility of RNA, its charged backbone, dearth of sp. surface features, and propensity for kinetic traps all conspire with its long folding time, to challenge in silico methods for physics-based folding. On the other hand, base-pairing interactions (either in runs to form helixes or isolated tertiary contacts) and motifs are often available from relatively low-cost expts. or informatics analyses. We present RNABuilder, a novel code that uses internal coordinate mechanics to satisfy user-specified base pairing and steric forces under chem. constraints. The code recapitulates the topol. and characteristic L-shape of tRNA and obtains an accurate noncrystallog. structure of the Tetrahymena ribozyme P4/P6 domain. The algorithm scales nearly linearly with mol. size, opening the door to the modeling of significantly larger structures.
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    Flores, S. C.; Bernauer, J.; Shin, S.; Zhou, R.; Huang, X. Multiscale Modeling of Macromolecular Biosystems. Brief. Bioinform. 2012, 13 (4), 395– 405,  DOI: 10.1093/bib/bbr077
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    Multiscale modeling of macromolecular biosystems
    Flores, Samuel C.; Bernauer, Julie; Shin, Seokmin; Zhou, Ruhong; Huang, Xuhui
    Briefings in Bioinformatics (2012), 13 (4), 395-405CODEN: BBIMFX; ISSN:1467-5463. (Oxford University Press)
    In this article, we review the recent progress in multiresoln. modeling of structure and dynamics of protein, RNA and their complexes. Many approaches using both physics-based and knowledge-based potentials have been developed at multiple granularities to model both protein and RNA. Coarse graining can be achieved not only in the length, but also in the time domain using discrete time and discrete state kinetic network models. Models with different resolns. can be combined either in a sequential or parallel fashion. Similarly, the modeling of assemblies is also often achieved using multiple granularities. The progress shows that a multiresoln. approach has considerable potential to continue extending the length and time scales of macromol. modeling.
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    Černý, J.; Božíková, P.; Svoboda, J.; Schneider, B. A Unified Dinucleotide Alphabet Describing Both RNA and DNA Structures. Nucleic Acids Res. 2020, 48 (11), 6367– 6381,  DOI: 10.1093/nar/gkaa383
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    A unified dinucleotide alphabet describing both RNA and DNA structures
    Cerny, Jiri; Bozikova, Paulina; Svoboda, Jakub; Schneider, Bohdan
    Nucleic Acids Research (2020), 48 (11), 6367-6381CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    By analyzing almost 120 000 dinucleotides in over 2000 nonredundant nucleic acid crystal structures, we define 96+1 diNucleotide Conformers, NtCs, which describe the geometry of RNA and DNA dinucleotides. NtC classes are grouped into 15 codes of the structural alphabet CANA (Conformational Alphabet of Nucleic Acids) to simplify symbolic annotation of the prominent structural features of NAs and their intuitive graphical display. The search for nontrivial patterns of NtCs resulted in the identification of several types of RNA loops, some of them obsd. for the first time. Over 30% of the nearly six million dinucleotides in the PDB cannot be assigned to any NtC class but we demonstrate that up to a half of them can be re-refined with the help of proper refinement targets. A statistical anal. of the preferences of NtCs and CANA codes for the 16 dinucleotide sequences showed that neither the NtC class AA00, which forms the scaffold of RNA structures, nor BB00, the DNA most populated class, are sequence neutral but their distributions are significantly biased. The reported automated assignment of the NtC classes and CANA codes available at dnatco.org provides a powerful tool for unbiased anal. of nucleic acid structures by structural and mol. biologists.
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    Černý, J.; Božíková, P.; Malý, M.; Tykač, M.; Biedermannová, L.; Schneider, B. Structural Alphabets for Conformational Analysis of Nucleic Acids Available at Dnatco.Datmos.Org. Acta Crystallogr. Sect. D Struct. Biol. 2020, 76 (9), 805– 813,  DOI: 10.1107/S2059798320009389
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    Structural alphabets for conformational analysis of nucleic acids available at dnatco.datmos.org
    Cerny, Jiri; Bozikova, Paulina; Maly, Michal; Tykac, Michal; Biedermannova, Lada; Schneider, Bohdan
    Acta Crystallographica, Section D: Structural Biology (2020), 76 (9), 805-813CODEN: ACSDAD; ISSN:2059-7983. (International Union of Crystallography)
    A detailed description of the dnatco.datmos.org web server implementing the universal structural alphabet of nucleic acids is presented. It is capable of processing any mmCIF- or PDB-formatted files contg. DNA or RNA mols.; these can either be uploaded by the user or supplied as the wwPDB or PDB-REDO structural database access code. The web server performs an assignment of the nucleic acid conformations and presents the results for the intuitive annotation, validation, modeling and refinement of nucleic acids.
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    Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E. GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1–2, 19– 25,  DOI: 10.1016/j.softx.2015.06.001
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    Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from Ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
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    ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
    Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
    Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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    Liebl, K.; Zacharias, M. Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry. J. Chem. Theory Comput. 2021, 17 (11), 7096– 7105,  DOI: 10.1021/acs.jctc.1c00682
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    Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry
    Liebl, Korbinian; Zacharias, Martin
    Journal of Chemical Theory and Computation (2021), 17 (11), 7096-7105CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    An accurate mol. mechanics force field forms the basis of Mol. Dynamics simulations to obtain a realistic view of the structure and dynamics of biomols. such as DNA. Although frequently updated to improve agreement with available exptl. data, DNA force fields still rely in part on parameters introduced more than 20 years ago. We have developed an entirely new DNA force field, Tumuc1, derived from quantum mech. calcns. to obtain a consistent set of bonded parameters and partial at. charges. The performance of the force field was extensively tested on a variety of DNA mols. It excels in accuracy of B-DNA simulations but also performs very well on other types of DNA structures and structure formation processes such as hairpin folding, duplex formation, and dynamics of DNA-protein complexes. It can complement existing force fields in order to provide an increasingly accurate description of the structure and dynamics of DNA during simulation studies.
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    Xu, G.; Chance, M. R. Radiolytic Modification and Reactivity of Amino Acid Residues Serving as Structural Probes for Protein Footprinting. Anal. Chem. 2005, 77 (14), 4549– 4555,  DOI: 10.1021/ac050299+
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    Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting
    Xu, Guozhong; Chance, Mark R.
    Analytical Chemistry (2005), 77 (14), 4549-4555CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examg. the structure and dynamics of biol. assemblies. In this study, the reactivities and tendencies to form easily detectible products for all 20 (common) amino acid side chains along with cystine are directly compared using various stds. Although we have previously reported on the oxidn. of many of these residues, this study includes a detailed examn. of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting expts. All 20 amino amides along with cystine and a few tripeptides were irradiated by γ-rays, the products were analyzed by electrospray mass spectrometry, and rate consts. of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal stds. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical expts. and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidn. products are not easily detectible. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical expts. Since these residues comprise ∼65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.
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    Niu, B.; Gross, M. L. MS-Based Hydroxyl Radical Footprinting: Methodology and Application of Fast Photochemical Oxidation of Proteins (FPOP). In Mass Spectrometry-Based Chemical Proteomics; Wiley, 2019; pp 363– 416.  DOI: 10.1002/9781118970195.ch15 .
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    Yin, V.; Mian, S. H.; Konermann, L. Lysine Carbonylation Is a Previously Unrecognized Contributor to Peroxidase Activation of Cytochrome c by Chloramine-T. Chem. Sci. 2019, 10 (8), 2349– 2359,  DOI: 10.1039/C8SC03624A
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    Lysine carbonylation is a previously unrecognized contributor to peroxidase activation of cytochrome c by chloramine-T
    Yin, Victor; Mian, Safee H.; Konermann, Lars
    Chemical Science (2019), 10 (8), 2349-2359CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    The peroxidase activity of cytochrome c (cyt c) plays a key role during apoptosis. Peroxidase catalysis requires a vacant Fe coordination site, i.e., cyt c must undergo an activation process involving structural changes that rupture the native Met80-Fe contact. A common strategy for dissocg. this bond is the conversion of Met80 to sulfoxide (MetO). It is widely believed that this MetO formation in itself is sufficient for cyt c activation. This notion originates from studies on chloramine-T-treated cyt c (CT-cyt c) which represents a std.model for the peroxidase activated state. CT-cyt c is considered to be a "clean" species that has undergone selective MetO formation, without any other modifications. Using optical, chromatog., and mass spectrometry techniques, the current work demonstrates that CT-induced activation of cyt c is more complicated than previously thought. MetO formation alone results in only marginal peroxidase activity, because dissocn. of the Met80-Fe bond triggers alternative ligation scenarios where Lys residues interfere with access to the heme. We found that CT causes not only MetO formation, but also carbonylation of several Lys residues. Carbonylation is assocd. with -1 Da mass shifts that have gone undetected in the CT-cyt c literature. Proteoforms possessing both MetO and Lys carbonylation exhibit almost fourfold higher peroxidase activity than those with MetO alone. Carbonylation abrogates the capability of Lys to coordinate the heme, thereby freeing up the distal site as required for an active peroxidase. Previous studies on CT-cyt c may have inadvertently examd. carbonylated proteoforms, potentially misattributing effects of carbonylation to solely MetO formation.
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    Liu, X. R.; Zhang, M. M.; Gross, M. L. Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications. Chemical Reviews 2020, 4355– 4454,  DOI: 10.1021/acs.chemrev.9b00815
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    Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications
    Liu, Xiaoran Roger; Zhang, Mengru Mira; Gross, Michael L.
    Chemical Reviews (Washington, DC, United States) (2020), 120 (10), 4355-4454CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Proteins adopt different higher-order structures (HOS) to enable their unique biol. functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS detn. of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS anal., through which different covalent labeling approaches "mark" the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen-deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resoln., the authors present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biol. questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a ref. for investigators seeking a MS-based tool to address structural questions in protein science.
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    Boura, E.; Silhan, J.; Herman, P.; Vecer, J.; Sulc, M.; Teisinger, J.; Obsilova, V.; Obsil, T. Both the N-Terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 Are Important for DNA Binding. J. Biol. Chem. 2007, 282 (11), 8265– 8275,  DOI: 10.1074/jbc.M605682200
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    Both the N-terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 are Important for DNA Binding
    Boura, Evzen; Silhan, Jan; Herman, Petr; Vecer, Jaroslav; Sulc, Miroslav; Teisinger, Jan; Obsilova, Veronika; Obsil, Tomas
    Journal of Biological Chemistry (2007), 282 (11), 8265-8275CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    FoxO4 belongs to the "O" subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel β-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors. In this report, we investigated the role of the N-terminal loop (portion located upstream of first helix H1) and the C-terminal region (loop known as wing W2) of the forkhead domain of transcription factor FoxO4 in DNA binding. Although the deletion of either portion partly reduces the FoxO4-DBD binding to the DNA, the simultaneous deletion of both regions inhibits DNA binding significantly. Foerster resonance energy transfer measurements and mol. dynamics simulations suggest that both studied N- and C-terminal regions of FoxO4-DBD directly interact with DNA. In the presence of the N-terminal loop the protein kinase B-induced phosphorylation of wing W2 by itself has negligible effect on DNA binding. On the other hand, in the absence of this loop the phosphorylation of wing W2 significantly inhibits the FoxO4-DBD binding to the DNA. The binding of the 14-3-3 protein efficiently reduces DNA-binding potential of phosphorylated FoxO4-DBD regardless of the presence of the N-terminal loop. Our results show that both N- and C-terminal regions of forkhead domain are important for stability of the FoxO4-DBD·DNA complex.
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    Obsilova, V.; Vecer, J.; Herman, P.; Pabianova, A.; Sulc, M.; Teisinger, J.; Boura, E.; Obsil, T. 14–3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4. Biochemistry 2005, 44 (34), 11608– 11617,  DOI: 10.1021/bi050618r
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    14-3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4
    Obsilova, Veronika; Vecer, Jaroslav; Herman, Petr; Pabianova, Anna; Sulc, Miroslav; Teisinger, Jan; Boura, Evzen; Obsil, Tomas
    Biochemistry (2005), 44 (34), 11608-11617CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The 14-3-3 proteins are a family of regulatory signaling mols. that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 Proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of its nuclear localization sequence (NLS). Masking or obscuring of the NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equil. of FoxO localization toward the cytoplasm. To the best of our knowledge, there is no exptl. evidence showing a direct interaction between the 14-3-3 protein and the NLS of FoxO. Therefore, the main goal of this work was to investigate whether phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of the FoxO4 NLS. We have used site-directed labeling of the FoxO4 NLS with the extrinsic fluorophore 1,5-IAEDANS in conjunction with steady-state and time-resolved fluorescence spectroscopy to study conformational changes of the FoxO4 NLS in vitro. Our data show that 14-3-3 protein binding significantly changes the environment around the AEDANS-labeled NLS and reduces its flexibility. On the other hand, phosphorylation itself and the binding of double-stranded DNA have a small effect on the structure of this region. Our results also suggest that the DNA-binding domain of FoxO4 remains relatively mobile while bound to the 14-3-3 protein.
  65. 65
    James, V. K.; Sanders, J. D.; Aizikov, K.; Fort, K. L.; Grinfeld, D.; Makarov, A.; Brodbelt, J. S. Advancing Orbitrap Measurements of Collision Cross Sections to Multiple Species for Broad Applications. Anal. Chem. 2022, 94 (45), 15613– 15620,  DOI: 10.1021/acs.analchem.2c02146
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    Advancing Orbitrap Measurements of Collision Cross Sections to Multiple Species for Broad Applications
    James, Virginia K.; Sanders, James D.; Aizikov, Konstantin; Fort, Kyle L.; Grinfeld, Dmitry; Makarov, Alexander; Brodbelt, Jennifer S.
    Analytical Chemistry (Washington, DC, United States) (2022), 94 (45), 15613-15620CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Measurement of collision cross section (CCS), a parameter reflecting an ion's size and shape, alongside high-resoln. mass anal. extends the depth of mol. anal. by providing structural information beyond mol. mass alone. Although these measurements are most commonly undertaken using a dedicated ion mobility cell coupled to a mass spectrometer, alternative methods have emerged to ext. CCSs directly by anal. of the decay rates of either time-domain transient signals or the FWHM of frequency domain peaks in FT mass analyzers. This information is also accessible from FTMS mass spectra obtained in commonly used workflows directly without the explicit access to transient or complex Fourier spectra. Previously, these expts. required isolation of individual charge states of ions prior to CCS anal., limiting throughput. Here we advance Orbitrap CCS measurements to more users and applications by detg. CCSs from commonly available mass spectra files as well as estg. CCS for multiple charge states simultaneously and showcase these methods by the measurement of CCSs of fragment ions produced from collisional activation of proteins.

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  1. Marek Zakopcanik, Daniel Kavan, Zdenek Kukacka, Petr Novak, Dmitry S. Loginov. Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis. Analytical Chemistry 2024, 96 (28) , 11273-11279. https://doi.org/10.1021/acs.analchem.4c01084
  2. Nicholas B. Borotto. The path forward for protein footprinting, covalent labeling, and mass spectrometry‐based protein conformational analyses. Journal of Mass Spectrometry 2024, 59 (7) https://doi.org/10.1002/jms.5064
  3. Clinton G.L. Veale, David J. Clarke. Mass spectrometry-based methods for characterizing transient protein–protein interactions. Trends in Chemistry 2024, 6 (7) , 377-391. https://doi.org/10.1016/j.trechm.2024.05.002
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  • Abstract

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    Figure 1

    Figure 1. Zoomed mass spectrum on a +14-charge state (m/z 842–851) showing an isotopic distribution of isotopically natural (IN-, A) and isotopically depleted (ID-, D) FOXO4-DBD. Fast photochemical oxidation of IN-FOXO4 without (B) and with (C) dsIRE. Fast photochemical oxidation of ID-FOXO4 without (E) and with (F) dsIRE.

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    Figure 2

    Figure 2. Histograms displaying the number of quantified fragment ions generated by ECD fragmentation of singly oxidized precursor ions of IN-FOXO4-DBD and ID-FOXO4-DBD.

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    Figure 3

    Figure 3. Zoomed ECD spectrum obtained upon fragmentation of isotopically natural (A) and isotopically depleted (B) FOXO4-DBD. The [c21]2+ is indicated with blue asterisks; the low-abundance [c57]6+ fragment ion is denoted by green squares; and its oxidized form, [c57+O]6+, is indicated by pink dots. The oxidized fragment ion [z38+O]4 is denoted by magenta triangles.

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    Figure 4

    Figure 4. MS/MS spectrum zoomed in the m/z range 1008–1012.300. The control ECD spectrum of unmodified ID-FOXO4-DBD is colored in black in the top panel (A). The ECD spectrum of oxidized ID-FOXO4-DBD with (B) and without IRE (C) is colored in blue and in red. The isotopic distribution of both [c73]8+ and [c73+O]8+fragment ions is denoted by transparent asterisks. Yellow dots denote lysine carbonylation within the protein, represented by the loss of 1.013 Da, while the green dots represent the oxidation of protein to its keto form (+13.9793). An ECD MS/MS spectrum of IN-FOXO4-DBD without IRE (D) and with IRE (E) shows no visible lysine carbonylation or oxidation to keto form.

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    Figure 5

    Figure 5. Plots indicating changes in oxidation rates between apo and holo forms of isotopically natural FOXO4 (A) and isotopically depleted FOXO4 (B); assessed by ECD fragmentation in multiCASI mode (Figure S5, Figure S8). Blue histograms represent changes in which region/residue was protected by IRE, and red histograms represent changes which resulted in deprotection of region/residue by IRE. (C) Changes obtained in ID-FOXO4-DBD were visualized into the differential oxidation map of FOXO4-DBD. The bold sequence represents spatial resolution achieved by fragmentation of isotopically depleted FOXO4-DBD. Colored residues were also detected by bottom-up analysis, as shown in Figure S11B and Table S1.

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    Figure 6

    Figure 6. An in silico structural model of FOXO4-DBD·IRE (PDB template 3L2C) (22) with the highlighted differently oxidized regions/residues detected by both top-down analyses for natural version (A) or depleted version (B) of FOXO4-DBD. The individual residues detected in either bottom-up approach or deduced from top-down were highlighted in the model and colored. Blue: regions/residues detected as more modified in apo form; red: regions/residues detected as more modified in holo form.

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      The use of mass spectrometry to study protein-ligand interactions is expanding into more complex systems including protein-DNA interactions. The excess amt. of a model DNA or, more typically, an oligodeoxynucleotide (ODN), needed to study such interactions in an amide hydrogen-deuterium (H/D) exchange expt., for example, causes serious signal suppression in the protein anal. We describe here a modification of the traditional H/D exchange protocol whereby we utilize a strong anion exchange column to rapidly remove the ODN from soln. before MS anal. We showed the successful incorporation of such a column in a study of two protein-ODN systems: (1) the DNA-binding domain of human telomeric repeat binding factor 2 with a telomeric oligodeoxynucleotide and (2) thrombin with the thrombin-binding aptamer. The approach gave no appreciable difference in back-exchange compared to a method in which no strong anion exchange (SAX) is used.
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      A New H/D Exchange- and Mass Spectrometry-Based Method for Thermodynamic Analysis of Protein-DNA Interactions
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      The application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the thermodn. anal. of protein-DNA complexes is described. A series of five model protein-DNA complexes involving two known DNA binding proteins, Arc repressor and CopG, were analyzed in order to det. the accuracy, precision, and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions. For protein-DNA complexes that reversibly unfold in a two-state manner, we demonstrate that reasonably precise Kd values in agreement with those detd. by conventional techniques can be detd. by SUPREX. In the case of protein-DNA complexes that are not well modeled by a two-state unfolding mechanism, we find that relative binding affinities can be detd. in the SUPREX expt.
    12. 12
      Sperry, J. B.; Shi, X.; Rempel, D. L.; Nishimura, Y.; Akashi, S.; Gross, M. L. A Mass Spectrometric Approach to the Study of DNA-Binding Proteins: Interaction of Human TRF2 with Telomeric DNA. Biochemistry 2008, 47 (6), 1797– 1807,  DOI: 10.1021/bi702037p
      12
      A Mass Spectrometric Approach to the Study of DNA-Binding Proteins: Interaction of Human TRF2 with Telomeric DNA
      Sperry, Justin B.; Shi, Xiangguo; Rempel, Don L.; Nishimura, Yoshifumi; Akashi, Satoko; Gross, Michael L.
      Biochemistry (2008), 47 (6), 1797-1807CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Human telomeric repeat binding factor 2 (hTRF2) is a protein that plays an important role in capping human telomeres to protect them from DNA damage repair systems. The ineffectiveness of hTRF2 may be linked to aging and cancer. We report the use of PLIMSTEX (protein-ligand interactions by mass spectrometry, titrn., and H/D exchange) and selective acetylation of lysine residues to study the interaction of the DNA-binding domain and double-stranded telomeric DNA (repeats of TTAGGG). By increasing the resoln. of PLIMSTEX to the peptide level, we localized the changes in deuterium uptake of hTRF2 as a function of varying amts. of a model oligodeoxynucleotide. From these expts., we detd. the affinity const. for binding to DNA, which is within a factor of 3 of the previously reported value. Amide H/D exchange revealed portions of the protein that have contacts with the phosphate backbone of DNA, whereas acetylation disclosed the decrease in solvent accessibility of regions contg. Lys 447 and 488, which must be involved in interactions with the DNA major and minor grooves. These complementary approaches of amide H/D exchange and selective side chain modification can be employed effectively to pinpoint and quantify protein-ligand, in particular protein-DNA, interactions.
    13. 13
      Gau, B. C.; Chen, H.; Zhang, Y.; Gross, M. L. Sulfate Radical Anion as a New Reagent for Fast Photochemical Oxidation of Proteins. Anal. Chem. 2010, 82 (18), 7821– 7827,  DOI: 10.1021/ac101760y
      13
      Sulfate Radical Anion as a New Reagent for Fast Photochemical Oxidation of Proteins
      Gau, Brian C.; Chen, Hao; Zhang, Yun; Gross, Michael L.
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      The focus is to expand the original design of fast photochem. oxidn. of proteins (FPOP) and introduce SO4-·, generated by 248 nm homolysis of low millimolar levels of persulfate, as a radical reactant in protein footprinting. FPOP is a chem. approach to footprinting proteins and protein complexes by "snapshot" reaction with free radicals. The radical used until now is the OH radical, and it provides a measure of residue-resolved solvent accessibility of the native protein. The authors show that FPOP can accommodate other reagents, increasing its versatility. The new persulfate FPOP system is a potent, nonspecific, and tunable footprinting method; 3-5 times less persulfate is needed to give the same global levels of modification as seen with OH radicals. Although solvent-exposed His and Tyr residues are more reactive with SO4-· than with ·OH, oxidn. of apomyoglobin and calmodulin shows that ·OH probes smaller accessible areas than SO4-·, with the possible exception of histidine. His64, an axial ligand in the heme-binding pocket of apomyoglobin, is substantially up-labeled by SO4-· relative to ·OH. Nevertheless, the kinds of modification and residue selectivity for both reagent radicals are strikingly similar. Thus, the choice of these reagents relies on the phys. properties, particularly the membrane permeability, of the radical precursors.
    14. 14
      Chen, J.; Cui, W.; Giblin, D.; Gross, M. L. New Protein Footprinting: Fast Photochemical Iodination Combined with Top-Down and Bottom-Up Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2012, 23 (8), 1306– 1318,  DOI: 10.1007/s13361-012-0403-1
      14
      New Protein Footprinting: Fast Photochemical Iodination Combined with Top-Down and Bottom-Up Mass Spectrometry
      Chen, Jiawei; Cui, Weidong; Giblin, Daryl; Gross, Michael L.
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      We report a new approach for the fast photochem. oxidn. of proteins (FPOP) whereby iodine species are used as the modifying reagent. We generate the radicals by photolysis of iodobenzoic acid at 248 nm; the putative iodine radical then rapidly modifies the target protein. This iodine-radical labeling is sensitive, tunable, and site-specific, modifying only histidine and tyrosine residues in contrast to OH radicals that modify 14 amino-acid side chains. We iodinated myoglobin (Mb) and apomyoglobin (aMb) in their native states and analyzed the outcome by both top-down and bottom-up proteomic strategies. Top-down sequencing selects a certain level (addn. of one I, two I's) of modification and dets. the major components produced in the modification reaction, whereas bottom-up reveals details for each modification site. Tyr146 is found to be modified for aMb but less so for Mb. His82, His93, and His97 are at least 10 times more modified for aMb than for Mb, in agreement with NMR studies. For carbonic anhydrase and its apo form, there are no significant differences of the modification extents, indicating their similarity in conformation and providing a control for this approach. For lispro insulin, insulin-EDTA, and insulin complexed with zinc, iodination yields are sensitive to differences in insulin oligomerization state. The iodine radical labeling is a promising addn. to protein footprinting methods, offering higher specificity and lower reactivity than ·OH and SO, two other radicals already employed in FPOP.
    15. 15
      Manzi, L.; Barrow, A. S.; Hopper, J. T. S.; Kaminska, R.; Kleanthous, C.; Robinson, C. V.; Moses, J. E.; Oldham, N. J. Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein. Angew. Chemie Int. Ed. 2017, 56 (47), 14873– 14877,  DOI: 10.1002/anie.201708254
      15
      Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein
      Manzi, Lucio; Barrow, Andrew S.; Hopper, Jonathan T. S.; Kaminska, Renata; Kleanthous, Colin; Robinson, Carol V.; Moses, John E.; Oldham, Neil J.
      Angewandte Chemie, International Edition (2017), 56 (47), 14873-14877CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Mapping the interaction sites between membrane-spanning proteins is a key challenge in structural biol. A carbene-footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilized in micelles. The diazirine-based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labeling of the membrane-spanning regions of the protein upon irradn. at 349 nm. Areas assocd. with protein-protein interactions between the trimer subunits remained unlabeled, thus revealing their location.
    16. 16
      Zhang, M. M.; Rempel, D. L.; Gross, M. L. A Fast Photochemical Oxidation of Proteins (FPOP) Platform for Free-Radical Reactions: The Carbonate Radical Anion with Peptides and Proteins. Free Radic. Biol. Med. 2019, 131, 126– 132,  DOI: 10.1016/j.freeradbiomed.2018.11.031
      16
      A Fast Photochemical Oxidation of Proteins (FPOP) platform for free-radical reactions: the carbonate radical anion with peptides and proteins
      Zhang, Mengru Mira; Rempel, Don L.; Gross, Michael L.
      Free Radical Biology & Medicine (2019), 131 (), 126-132CODEN: FRBMEH; ISSN:0891-5849. (Elsevier B.V.)
      Fast Photochem. Oxidn. of Protein (FPOP), based on a pulsed KrF laser (248 nm) for free-radical generation, is a biophys. method that utilizes hydroxyl radicals to footprint proteins in soln. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity detn. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast ("snapshot") footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradn. of small plugs of the targeted sample; iii) the use of methionine and catalase after radical oxidn. chem. to prevent post-oxidn. with residual oxidizing species; and iv) the utilization of mature mass spectrometry-based proteomic methods to afford detailed anal. In addn. to •OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biol. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO-•3. Other arom. amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliph. amino acids are inert, unlike with •OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.
    17. 17
      Cheng, M.; Zhang, B.; Cui, W.; Gross, M. L. Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting. Angew. Chemie - Int. Ed. 2017, 56 (45), 14007– 14010,  DOI: 10.1002/anie.201706697
      17
      Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting
      Cheng, Ming; Zhang, Bojie; Cui, Weidong; Gross, Michael L.
      Angewandte Chemie, International Edition (2017), 56 (45), 14007-14010CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Described is a novel, laser-initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. *CF3 reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to .OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chem. and structural biol. and biotechnol. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.
    18. 18
      Fojtík, L.; Fiala, J.; Pompach, P.; Chmelík, J.; Matoušek, V.; Beier, P.; Kukačka, Z.; Novák, P. Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules. J. Am. Chem. Soc. 2021, 143 (49), 20670– 20679,  DOI: 10.1021/jacs.1c07771
      18
      Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules
      Fojtik, Lukas; Fiala, Jan; Pompach, Petr; Chmelik, Josef; Matousek, Vaclav; Beier, Petr; Kukacka, Zdenek; Novak, Petr
      Journal of the American Chemical Society (2021), 143 (49), 20670-20679CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure detn. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resoln. to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decompn. of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-Hb complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.
    19. 19
      Sharp, J. S.; Becker, J. M.; Hettich, R. L. Protein Surface Mapping by Chemical Oxidation: Structural Analysis by Mass Spectrometry. Anal. Biochem. 2003, 313 (2), 216– 225,  DOI: 10.1016/S0003-2697(02)00612-7
      19
      Protein surface mapping by chemical oxidation: Structural analysis by mass spectrometry
      Sharp, Joshua S.; Becker, Jeffrey M.; Hettich, Robert L.
      Analytical Biochemistry (2003), 313 (2), 216-225CODEN: ANBCA2; ISSN:0003-2697. (Elsevier Science)
      The solvent-accessible surface area of proteins is important in biol. function for many reasons, including protein-protein interactions, protein folding, and catalytic sites. Here we present a chem. technique to oxidize amino acid side chains in a model protein, apomyoglobin, and subsequent elucidation of the effect of solvent accessibility on the sites of oxidn. Under conditions of low protein oxidn. (zero to three oxygen atoms added per apomyoglobin mol.), we have pos. identified five oxidn. sites by liq. chromatog.-tandem mass spectrometry and high-resoln. Fourier transform mass spectrometry. Our results indicate that all oxidized amino acids, with the exception of methionine, have highly solvent-accessible side chains, but the rate of oxidn. may not be dictated solely by solvent accessibility and amino acid identity.
    20. 20
      Hambly, D. M.; Gross, M. L. Laser Flash Photolysis of Hydrogen Peroxide to Oxidize Protein Solvent-Accessible Residues on the Microsecond Timescale. J. Am. Soc. Mass Spectrom. 2005, 16 (12), 2057– 2063,  DOI: 10.1016/j.jasms.2005.09.008
      20
      Laser Flash Photolysis of Hydrogen Peroxide to Oxidize Protein Solvent-Accessible Residues on the Microsecond Timescale
      Hambly, David M.; Gross, Michael L.
      Journal of the American Society for Mass Spectrometry (2005), 16 (12), 2057-2063CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      Footprinting of proteins by hydroxyl radicals generated on the millisecond to minute timescales to probe protein surfaces suffers from the uncertainty that radical reactions cause the protein to unfold, exposing residues that are protected in the native protein. To circumvent this possibility, the authors developed a method using a 248 nm KrF excimer laser to cleave hydrogen peroxide at low concns. (15 mM, 0.04%), affording hydroxyl radicals that modify the protein in less than a microsecond. In the presence of a scavenger (20 mM glutamine), the radical lifetimes decrease to ∼1 μs, yet the reaction timescales are sufficient to provide significant oxidn. of the protein. These times are arguably faster than super-secondary protein structure can unfold as a result of the modification. The radical formation step takes place in a nanoliter flow cell so that only one laser pulse irradiates each bolus of sample. The oxidn. sites are located using std. anal. proteomics, requiring less than a nanomole of protein. The authors tested the method with apomyoglobin and obsd. modifications in accord with solvent accessibility data obtained from the crystal structure of holomyoglobin. Addnl., the results indicate that the F-helix is conformationally flexible in apomyoglobin, in accord with NMR results. The authors also find that the binding pocket is resistant to modifications, indicating that the protein pocket closes in the absence of the heme group - conclusions that cannot be drawn from current structural methods. When developed further, this method may enable the detn. of protein-ligand interfaces, affinity consts., folding pathways, and regions of conformational flexibility.
    21. 21
      Wang, L.; Chance, M. R. Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment. Mol. Cell. Proteomics 2017, 16 (5), 706– 716,  DOI: 10.1074/mcp.O116.064386
      21
      Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment
      Wang, Liwen; Chance, Mark R.
      Molecular & Cellular Proteomics (2017), 16 (5), 706-716CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
      Protein footprinting mediated by mass spectrometry has evolved over the last 30 years from proof of concept to commonplace biophysics tool, with unique capabilities for assessing structure and dynamics of purified proteins in physiol. states in soln. This review outlines the history and current capabilities of two major methods of protein footprinting: reversible hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (HRF), an irreversible covalent labeling approach. Technol. advances in both approaches now permit high-resoln. assessments of protein structure including secondary and tertiary structure stability mediated by backbone interactions (measured via HDX) and solvent accessibility of side chains (measured via HRF). Applications across many academic fields and in biotechnol. drug development are illustrated including: detection of protein interfaces, identification of ligand/drug binding sites, and monitoring dynamics of protein conformational changes along with future prospects for advancement of protein footprinting in structural biol. and biophysics research.
    22. 22
      Liu, X. R.; Zhang, M. M.; Gross, M. L. Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications. Chem. Rev. 2020, 120 (10), 4355– 4454,  DOI: 10.1021/acs.chemrev.9b00815
      22
      Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications
      Liu, Xiaoran Roger; Zhang, Mengru Mira; Gross, Michael L.
      Chemical Reviews (Washington, DC, United States) (2020), 120 (10), 4355-4454CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Proteins adopt different higher-order structures (HOS) to enable their unique biol. functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS detn. of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS anal., through which different covalent labeling approaches "mark" the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen-deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resoln., the authors present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biol. questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a ref. for investigators seeking a MS-based tool to address structural questions in protein science.
    23. 23
      Xu, G.; Chance, M. R. Hydroxyl Radical-Mediated Modification of Proteins as Probes for Structural Proteomics. Chem. Rev. 2007, 107 (8), 3514– 3543,  DOI: 10.1021/cr0682047
      23
      Hydroxyl Radical-Mediated Modification of Proteins as Probes for Structural Proteomics
      Xu, Guozhong; Chance, Mark R.
      Chemical Reviews (Washington, DC, United States) (2007), 107 (8), 3514-3543CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review including major sections on background and history of protein footprinting, generation of hydroxy radicals in soln., hydroxy radical mediated cleavage of the main chain and modification of side chains, and future prospects.
    24. 24
      Xu, G.; Chance, M. R. Radiolytic Modification of Acidic Amino Acid Residues in Peptides: Probes for Examining Protein–Protein Interactions. Anal. Chem. 2004, 76 (5), 1213– 1221,  DOI: 10.1021/ac035422g
      24
      Radiolytic Modification of Acidic Amino Acid Residues in Peptides: Probes for Examining Protein-Protein Interactions
      Xu, Guozhong; Chance, Mark R.
      Analytical Chemistry (2004), 76 (5), 1213-1221CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Hydroxyl radical-mediated footprinting coupled with mass spectroscopic anal. is a new technique for mapping protein surfaces, identifying structural changes modulated by protein-ligand binding, and mapping protein-ligand interfaces in soln. In this study, the authors examine the radiolytic oxidn. of aspartic and glutamic acid residues to probe their potential use as structural probes in footprinting expts. Model peptides contg. Asp or Glu were irradiated using white light from a synchrotron x-ray source or a cesium-137 γ-ray source. The radiolysis products were characterized by electrospray mass spectrometry including tandem mass spectrometry. Both Asp and Glu are susceptible to radiolytic oxidization by γ-rays or synchrotron x-rays. Radiolysis results primarily in the oxidative decarboxylation of the side chain carboxyl group and formation of an aldehyde group at the carbon next to the original carboxyl group, giving rise to a characteristic product with a -30 Da mass change. A similar oxidative decarboxylation also takes place for amino acids with C-terminal carboxyl groups. The methylene groups in the Asp and Glu side chains also undergo oxygen addn. forming ketone or alc. groups with mass changes of +14 and +16 Da, resp. Characterizing the oxidn. reactions of these two acidic residues extends the no. of useful side chain probes for hydroxyl radical-mediated protein footprinting from 10 (Cys, Met, Trp, Tyr, Phe, Arg, Leu, Pro, His, Lys) to 12 amino acid residues, thus enhancing the ability to map protein surface structure, and in combination with previously identified basic amino acid probes, this method can be used to examine mol. details of protein-protein interactions that are driven by electrostatics.
    25. 25
      Xu, G.; Takamoto, K.; Chance, M. R. Radiolytic Modification of Basic Amino Acid Residues in Peptides: Probes for Examining Protein–Protein Interactions. Anal. Chem. 2003, 75 (24), 6995– 7007,  DOI: 10.1021/ac035104h
      25
      Radiolytic modification of basic amino acid residues in peptides: Probes for examining protein-protein interactions
      Xu, Guozhong; Takamoto, Keiji; Chance, Mark R.
      Analytical Chemistry (2003), 75 (24), 6995-7007CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Protein footprinting utilizing hydroxyl radicals coupled with mass spectrometry has become a powerful technique for mapping the solvent accessible surface of proteins and examg. protein-protein interactions in soln. Hydroxyl radicals generated by radiolysis or chem. methods efficiently react with many amino acid residue side chains, including the arom. and sulfur-contg. residues along with proline and leucine, generating stable oxidn. products that are valuable probes for examg. protein structure. In this study, we examine the radiolytic oxidn. chem. of histidine, lysine, and arginine for comparison with their metal-catalyzed oxidn. products. Model peptides contg. arginine, histidine, and lysine were irradiated using white light from a synchrotron x-ray source or a cesium-137 γ-ray source. The rates of oxidn. and the radiolysis products were primarily characterized by electrospray mass spectrometry including tandem mass spectrometry. Arginine is very sensitive to radiolytic oxidn., giving rise to a characteristic product with a 43 Da mass redn. as a result of the loss of guanidino group and conversion to γ-glutamyl semialdehyde, consistent with previous metal-catalyzed oxidn. studies. Histidine was oxidized to generate a mixt. of products with characteristic mass changes primarily involving rupture of and addn. to the imidazole ring. Lysine was converted to hydroxylysine or carbonylysine by radiolysis. The development of methods to probe these residues due to their high frequency of occurrence, their typical presence on the protein surface, and their frequent participation in protein-protein interactions considerably extends the utility of protein footprinting.
    26. 26
      Xu, G.; Chance, M. R. Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting. Anal. Chem. 2005, 77 (8), 2437– 2449,  DOI: 10.1021/ac0484629
      26
      Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting
      Xu, Guozhong; Chance, Mark R.
      Analytical Chemistry (2005), 77 (8), 2437-2449CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Protein footprinting based on hydroxyl radical-mediated modification and quant. mass spectroscopic anal. is a proven technique for examg. protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural anal. depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-contg. amino acids are oxidized readily and the mechanisms of oxidn. are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here the authors have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides contg. cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidn. using γ-rays or synchrotron x-rays and have compared these results to those expected from oxidn. mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are obsd. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidn. is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the γ-carbon. Due to the high reactivity of sulfur-contg. amino acids, the extent of oxidn. is easily influenced by secondary oxidn. events or the presence of redox reagents used in std. proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine ∼ tryptophan > cystine is obsd.
    27. 27
      Charvátová, O.; Foley, B. L.; Bern, M. W.; Sharp, J. S.; Orlando, R.; Woods, R. J. Quantifying Protein Interface Footprinting by Hydroxyl Radical Oxidation and Molecular Dynamics Simulation: Application to Galectin-1. J. Am. Soc. Mass Spectrom. 2008, 19 (11), 1692– 1705,  DOI: 10.1016/j.jasms.2008.07.013
      27
      Quantifying Protein Interface Footprinting by Hydroxyl Radical Oxidation and Molecular Dynamics Simulation: Application to Galectin-1
      Charvatova, Olga; Foley, B. Lachele; Bern, Marshall W.; Sharp, Joshua S.; Orlando, Ron; Woods, Robert J.
      Journal of the American Society for Mass Spectrometry (2008), 19 (11), 1692-1705CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      Biomol. surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to det. high-resoln. three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resoln. compared with traditional chem. derivatization. Here the authors present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissocn. of hydrogen peroxide. The authors applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient anal. of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large no. of modifications arising from side-chain oxidn. Quantification of the level of oxidn. has been achieved by employing spectral intensities for all of the obsd. oxidn. states on a per-residue basis. The level of accuracy achievable from spectral intensities was detd. by examn. of mixts. of synthetic peptides related to those present after oxidn. and tryptic digestion of galectin-1. A direct relation between side-chain solvent accessibility and level of oxidn. emerged, which enabled the prediction of the level of oxidn. given the 3D structure of the protein. The precision of this relation was enhanced through the use of av. solvent accessibilities computed from 10 ns mol. dynamics simulations of the protein.
    28. 28
      Pan, Y.; Stocks, B. B.; Brown, L.; Konermann, L. Structural Characterization of an Integral Membrane Protein in Its Natural Lipid Environment by Oxidative Methionine Labeling and Mass Spectrometry. Anal. Chem. 2009, 81 (1), 28– 35,  DOI: 10.1021/ac8020449
      28
      Structural Characterization of an Integral Membrane Protein in Its Natural Lipid Environment by Oxidative Methionine Labeling and Mass Spectrometry
      Pan, Yan; Stocks, Bradley B.; Brown, Leonid; Konermann, Lars
      Analytical Chemistry (Washington, DC, United States) (2009), 81 (1), 28-35CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Membrane proteins represent formidable challenges for many anal. techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a nonnatural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-contg. purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dil. aq. H2O2. The expts. rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following ·OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-sol. proteins, which generally undergo modifications at many different types of residues. In those earlier expts. the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidn. pattern obsd. here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidn. The authors' results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, esp. when considering that many of these species are methionine-rich. The introduction of addnl. Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodol. described here.
    29. 29
      Watkinson, T. G.; Calabrese, A. N.; Ault, J. R.; Radford, S. E.; Ashcroft, A. E. FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins. J. Am. Soc. Mass Spectrom. 2017, 28 (1), 50– 55,  DOI: 10.1007/s13361-016-1421-1
      29
      FPOP-LC-MS/MS Suggests Differences in Interaction Sites of Amphipols and Detergents with Outer Membrane Proteins
      Watkinson, Thomas G.; Calabrese, Antonio N.; Ault, James R.; Radford, Sheena E.; Ashcroft, Alison E.
      Journal of the American Society for Mass Spectrometry (2017), 28 (1), 50-55CODEN: JAMSEF; ISSN:1044-0305. (Springer)
      Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in soln., and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochem. oxidn. of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quant. mass spectrometry analyses, we show that fast photochem. oxidn. reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of addnl. intermol. contacts.
    30. 30
      Lu, Y.; Zhang, H.; Niedzwiedzki, D. M.; Jiang, J.; Blankenship, R. E.; Gross, M. L. Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc. Anal. Chem. 2016, 88 (17), 8827– 8834,  DOI: 10.1021/acs.analchem.6b01945
      30
      Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc
      Lu, Yue; Zhang, Hao; Niedzwiedzki, Dariusz M.; Jiang, Jing; Blankenship, Robert E.; Gross, Michael L.
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (17), 8827-8834CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Although membrane proteins are crucial participants in photosynthesis and other biol. processes, many lack high-resoln. structures. Prior to achieving a high-resoln. structure, the authors are studying whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. The authors' platform probes topol. and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochem. oxidn. of proteins (FPOP), and lipid Nanodiscs, which more similar to the native membrane environment than are the widely used detergent micelles. The authors describe here results that show a protein's outer membrane regions are more heavily footprinted by OH radicals whereas the regions spanning the lipid bilayer remain inert to the labeling. Nanodiscs generally exhibit more protection of membrane proteins compared to detergent micelles and less shielding to those protein residues that exist outside the membrane. The combination of immobilizing the protein in Nanodiscs and footprinting with the FPOP approach is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level, and to illuminate intrinsic membrane protein topol.
    31. 31
      Gupta, S.; Bavro, V. N.; D’Mello, R.; Tucker, S. J.; Vénien-Bryan, C.; Chance, M. R. Conformational Changes during the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry. Structure 2010, 18 (7), 839– 846,  DOI: 10.1016/j.str.2010.04.012
      31
      Conformational Changes During the Gating of a Potassium Channel Revealed by Structural Mass Spectrometry
      Gupta, Sayan; Bavro, Vassiliy N.; D'Mello, Rhijuta; Tucker, Stephen J.; Venien-Bryan, Catherine; Chance, Mark R.
      Structure (Cambridge, MA, United States) (2010), 18 (7), 839-846CODEN: STRUE6; ISSN:0969-2126. (Cell Press)
      Summary: Potassium channels are dynamic proteins that undergo large conformational changes to regulate the flow of K+ ions across the cell membrane. Understanding the gating mechanism of these channels therefore requires methods for probing channel structure in both their open and closed conformations. Radiolytic footprinting is used to study the gating mechanism of the inwardly-rectifying potassium channel KirBac3.1. The purified protein stabilized in either open or closed conformations was exposed to focused synchrotron X-ray beams on millisecond timescales to modify solvent accessible amino acid side chains. These modifications were identified and quantified using high-resoln. mass spectrometry. The differences obsd. between the closed and open states were then used to reveal local conformational changes that occur during channel gating. The results provide support for a proposed gating mechanism of the Kir channel and demonstrate a method of probing the dynamic gating mechanism of other integral membrane proteins and ion channels.
    32. 32
      Loginov, D. S.; Fiala, J.; Brechlin, P.; Kruppa, G.; Novak, P. Hydroxyl Radical Footprinting Analysis of a Human Haptoglobin-Hemoglobin Complex. Biochim. Biophys. Acta - Proteins Proteomics 2022, 1870 (2), 140735  DOI: 10.1016/j.bbapap.2021.140735
      32
      Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex
      Loginov, Dmitry S.; Fiala, Jan; Brechlin, Peter; Kruppa, Gary; Novak, Petr
      Biochimica et Biophysica Acta, Proteins and Proteomics (2022), 1870 (2), 140735CODEN: BBAPBW; ISSN:1570-9639. (Elsevier B.V.)
      Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochem. oxidn. of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile detn. of the site of modification with single residue resoln. In the present study, FPOP anal. was applied to study the Hb (Hb) - haptoglobin (Hp) complex allowing identification of resp. regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, resp., including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed detn. of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.
    33. 33
      Cornwell, O.; Bond, N. J.; Radford, S. E.; Ashcroft, A. E. Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS. Anal. Chem. 2019, 91 (23), 15163– 15170,  DOI: 10.1021/acs.analchem.9b03958
      33
      Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS
      Cornwell, Owen; Bond, Nicholas J.; Radford, Sheena E.; Ashcroft, Alison E.
      Analytical Chemistry (Washington, DC, United States) (2019), 91 (23), 15163-15170CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochem. Oxidn. of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labeling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal down-stream anal. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity detg. regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-assocn. at high concns. and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labeling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labeling patterns at the interfaces of the CL-CH1 and CH1-CH2 domains, with the nonaggregating variant undergoing up to four times more labeling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL - CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidized peptides. Some oxidized amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an anal. as an aid to MS/MS in assigning oxidn. sites.
    34. 34
      Cornwell, O.; Radford, S. E.; Ashcroft, A. E.; Ault, J. R. Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: A Structural Characterisation of Wild-Type and ΔN6 B2-Microglobulin. J. Am. Soc. Mass Spectrom. 2018, 29 (12), 2413– 2426,  DOI: 10.1007/s13361-018-2067-y
      34
      Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and ΔN6 β2-Microglobulin
      Cornwell, Owen; Radford, Sheena E.; Ashcroft, Alison E.; Ault, James R.
      Journal of the American Society for Mass Spectrometry (2018), 29 (12), 2413-2426CODEN: JAMSEF; ISSN:1044-0305. (Springer)
      Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochem. oxidn. of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β2-microglobulin (β2m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimized LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures. [Figure not available: see fulltext.].
    35. 35
      Yassaghi, G.; Kukačka, Z.; Fiala, J.; Kavan, D.; Halada, P.; Volný, M.; Novák, P. Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation. Anal. Chem. 2022, 94 (28), 9993– 10002,  DOI: 10.1021/acs.analchem.1c05476
      35
      Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation
      Yassaghi, Ghazaleh; Kukacka, Zdenek; Fiala, Jan; Kavan, Daniel; Halada, Petr; Volny, Michael; Novak, Petr
      Analytical Chemistry (Washington, DC, United States) (2022), 94 (28), 9993-10002CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Fast photochem. oxidn. of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chem. reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established std. method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixt. of peptides from various subpopulations of proteins with varying degrees of photochem. oxidn. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down expt. is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissocn., electron-transfer dissocn., and electron-capture dissocn. combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.
    36. 36
      Tomášková, N.; Novák, P.; Kožár, T.; Petrenčáková, M.; Jancura, D.; Yassaghi, G.; Man, P.; Sedlák, E. Early Modification of Cytochrome c by Hydrogen Peroxide Triggers Its Fast Degradation. Int. J. Biol. Macromol. 2021, 174, 413– 423,  DOI: 10.1016/j.ijbiomac.2021.01.189
      36
      Early modification of cytochrome c by hydrogen peroxide triggers its fast degradation
      Tomaskova, Natasa; Novak, Petr; Kozar, Tibor; Petrencakova, Martina; Jancura, Daniel; Yassaghi, Ghazaleh; Man, Petr; Sedlak, Erik
      International Journal of Biological Macromolecules (2021), 174 (), 413-423CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
      Cytochrome c (cyt c), in addn. to its function as an electron shuttle in respiratory chain, is able to perform as a pseudo-peroxidase with a crit. role during apoptosis. Incubation of cyt c with an excess of hydrogen peroxide leads to a suicide inactivation of the protein, which is accompanied by heme destruction and covalent modification of numerous amino acid residues. Although steady-state reactions of cyt c with an excess of hydrogen peroxide represent non-physiol. conditions, they might be used for anal. of the first-modified amino acid in in vivo. Here, we obsd. oxidn. of tyrosine residues 67 and 74 and heme as the first modifications found upon incubation with hydrogen peroxide. The positions of the oxidized tyrosines suggest a possible migration pathway of hydrogen peroxide-induced radicals from the site of heme localization to the protein surface. Anal. of a size of folded fraction of cyt c upon limited incubation with hydrogen peroxide indicates that the early oxidn. of amino acids triggers an accelerated destruction of cyt c. Position of channels from mol. dynamics simulation structures of cyt c points to a location of amino acid residues exposed to reactive oxidants that are thus more prone to covalent modification.
    37. 37
      Donnelly, D. P.; Rawlins, C. M.; DeHart, C. J.; Fornelli, L.; Schachner, L. F.; Lin, Z.; Lippens, J. L.; Aluri, K. C.; Sarin, R.; Chen, B.; Lantz, C.; Jung, W.; Johnson, K. R.; Koller, A.; Wolff, J. J.; Campuzano, I. D. G.; Auclair, J. R.; Ivanov, A. R.; Whitelegge, J. P.; Paša-Tolić, L.; Chamot-Rooke, J.; Danis, P. O.; Smith, L. M.; Tsybin, Y. O.; Loo, J. A.; Ge, Y.; Kelleher, N. L.; Agar, J. N. Best Practices and Benchmarks for Intact Protein Analysis for Top-down Mass Spectrometry. Nat. Methods 2019, 16 (7), 587– 594,  DOI: 10.1038/s41592-019-0457-0
      37
      Best practices and benchmarks for intact protein analysis for top-down mass spectrometry
      Donnelly, Daniel P.; Rawlins, Catherine M.; DeHart, Caroline J.; Fornelli, Luca; Schachner, Luis F.; Lin, Ziqing; Lippens, Jennifer L.; Aluri, Krishna C.; Sarin, Richa; Chen, Bifan; Lantz, Carter; Jung, Wonhyeuk; Johnson, Kendall R.; Koller, Antonius; Wolff, Jeremy J.; Campuzano, Iain D. G.; Auclair, Jared R.; Ivanov, Alexander R.; Whitelegge, Julian P.; Pasa-Tolic, Ljiljana; Chamot-Rooke, Julia; Danis, Paul O.; Smith, Lloyd M.; Tsybin, Yury O.; Loo, Joseph A.; Ge, Ying; Kelleher, Neil L.; Agar, Jeffrey N.
      Nature Methods (2019), 16 (7), 587-594CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic mol. mass. Top-down mass spectrometry enables the anal. of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass anal. of intact proteins (antibodies, membrane proteins and others) from mixts. of varying complexity. We also present cross-platform anal. benchmarks using a protein std. sample, to allow users to gauge their proficiency.
    38. 38
      Petrenčáková, M.; Filandr, F.; Hovan, A.; Yassaghi, G.; Man, P.; Kožár, T.; Schwer, M. S.; Jancura, D.; Plückthun, A.; Novák, P.; Miškovský, P.; Bánó, G.; Sedlák, E. Photoinduced Damage of AsLOV2 Domain Is Accompanied by Increased Singlet Oxygen Production Due to Flavin Dissociation. Sci. Rep. 2020, 10 (1), 1– 15,  DOI: 10.1038/s41598-020-60861-2
      There is no corresponding record for this reference.
    39. 39
      Kellersberger, K. A.; Yu, E.; Kruppa, G. H.; Young, M. M.; Fabris, D. Top-Down Characterization of Nucleic Acids Modified by Structural Probes Using High-Resolution Tandem Mass Spectrometry and Automated Data Interpretation. Anal. Chem. 2004, 76 (9), 2438– 2445,  DOI: 10.1021/ac0355045
      39
      Top-Down Characterization of Nucleic Acids Modified by Structural Probes Using High-Resolution Tandem Mass Spectrometry and Automated Data Interpretation
      Kellersberger, Katherine A.; Yu, Eizadora; Kruppa, Gary H.; Young, Malin M.; Fabris, Daniele
      Analytical Chemistry (2004), 76 (9), 2438-2445CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A top-down approach based on sustained off-resonance irradn. collision-induced dissocn. (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as di-Me sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and β-ethoxy-α-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chem. to produce strand cleavage at the modified nucleotides, and anal. by PAGE to det. the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the anal. of individual target adducts present in complex reaction mixts. with no need for sepn. techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MSn expts. to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated redn. and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data redn. module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calcs. the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data redn. program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on crosslinking strategies and top-down ESI-FTMS.
    40. 40
      Valkenborg, D.; Mertens, I.; Lemière, F.; Witters, E.; Burzykowski, T. The Isotopic Distribution Conundrum. Mass Spectrometry Reviews; John Wiley & Sons, Ltd, January 1, 2012; pp 96– 109.  DOI: 10.1002/mas.20339 .
      There is no corresponding record for this reference.
    41. 41
      Compton, P. D.; Zamdborg, L.; Thomas, P. M.; Kelleher, N. L. On the Scalability and Requirements of Whole Protein Mass Spectrometry. Anal. Chem. 2011, 83 (17), 6868– 6874,  DOI: 10.1021/ac2010795
      41
      On the Scalability and Requirements of Whole Protein Mass Spectrometry
      Compton, Philip D.; Zamdborg, Leonid; Thomas, Paul M.; Kelleher, Neil L.
      Analytical Chemistry (Washington, DC, United States) (2011), 83 (17), 6868-6874CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Top-down proteomics has improved over the past decade despite the significant challenges presented by the anal. of large protein ions. Here, the detection of these high mass species by electrospray-based mass spectrometry (MS) is examd. from a theor. perspective to understand the mass-dependent increases in the no. of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quant. model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of 13C and 15N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, the authors find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20-fold.
    42. 42
      Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S.; Guan, S.; Logan, T. M. Protein Molecular Mass to 1 Da by 13 C, 15 N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119 (2), 433– 434,  DOI: 10.1021/ja9630046
      42
      Protein Molecular Weight to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry
      Marshall, Alan G.; Senko, Michael W.; Li, Weiqun; Li, Ming; Dillon, Stephanie; Guan, Shenheng; Logan, Timothy M.
      Journal of the American Chemical Society (1997), 119 (2), 433-434CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Although electrosprayed protein gas-phase ion masses can be detd. to ppm accuracy, the broad natural-abundance isotopic distribution can lead to significant error (≥1 Da) in detn. of the protein monoisotopic mol. wt. Here, we show that a protein doubly-depleted in 13C and 15N exhibits a substantially narrowed isotopic distribution with greatly increased population of the monoisotopic species (all carbons are 12C, all nitrogens are 14N, all oxygens are 16O, etc.). The narrower isotopic distribution will facilitate MS/MS and adduct identification, and promises to extend substantially the upper limit for protein mass anal.
    43. 43
      Bou-Assaf, G. M.; Chamoun, J. E.; Emmett, M. R.; Fajer, P. G.; Marshall, A. G. Advantages of Isotopic Depletion of Proteins for Hydrogen/Deuterium Exchange Experiments Monitored by Mass Spectrometry. Anal. Chem. 2010, 82 (8), 3293– 3299,  DOI: 10.1021/ac100079z
      43
      Advantages of Isotopic Depletion of Proteins for Hydrogen/Deuterium Exchange Experiments Monitored by Mass Spectrometry
      Bou-Assaf, George M.; Chamoun, Jean E.; Emmett, Mark R.; Fajer, Piotr G.; Marshall, Alan G.
      Analytical Chemistry (Washington, DC, United States) (2010), 82 (8), 3293-3299CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Soln.-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein-protein interactions and conformational changes in biol. systems, esp. when traditional methods such as x-ray crystallog. or NMR are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein mol. wt.(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addn., different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of 13C and/or 15N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species (12Cc1Hh14Nn16Oo32Ss), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data redn.
    44. 44
      Charlebois, J. P.; Patrie, S. M.; Kelleher, N. L. Electron Capture Dissociation and 13C,15N Depletion for Deuterium Localization in Intact Proteins after Solution-Phase Exchange. Anal. Chem. 2003, 75 (13), 3263– 3266,  DOI: 10.1021/ac020690k
      44
      Electron Capture Dissociation and 13C, 15N Depletion for Deuterium Localization in Intact Proteins after Solution-Phase Exchange
      Charlebois, Jay P.; Patrie, Steven M.; Kelleher, Neil L.
      Analytical Chemistry (2003), 75 (13), 3263-3266CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      For localization of deuterium atoms after soln.-phase exchange with D2O, intact proteins are often digested prior to anal. by mass spectrometry (MS) and tandem MS (MS/MS). Amelioration of limitations assocd. with this approach (e.g., <70% sequence coverage and some D atom scrambling during MS/MS) were sought using intact proteins and two newer methods applied to tracking H/D exchange dynamics for the first time. Using 2-4-fold signal enhancements through depletion of 13C and 15N isotopes and implementing the new MS/MS technique of electron capture dissocn. (ECD) yielded an increased no. of c and z• ions obsd. (43 vs. 25) for recombinant yeast ubiquitin (9.3 kDa). Initial detn. of D atom content in consecutive c ion series (c4 - c7, c28, c31, c32, and c33) was demonstrated. The improved ion signal and expt. speed combined with narrower isotopic distributions markedly increases the degree of localization and feasibility of ECD-based MS/MS after soln.-phase H/D exchange.
    45. 45
      Zubarev, R. A.; Demirev, P. A. Isotope Depletion of Large Biomolecules: Implications for Molecular Mass Measurements. J. Am. Soc. Mass Spectrom. 1998, 9 (2), 149– 156,  DOI: 10.1016/S1044-0305(97)00232-8
      45
      Isotope depletion of large biomolecules: implications for molecular mass measurements
      Zubarev, Roman A.; Demirev, Plamen A.
      Journal of the American Society for Mass Spectrometry (1998), 9 (2), 149-156CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Science Inc.)
      Isotope depletion (or enrichment) of large biomols. is a procedure already used in high resoln. Fourier transform ion cyclotron resonance mass spectrometry for improving the reliability and accuracy of biomol. mass characterization. In this work, effects of isotope depletion on a no. of mass spectrometric parameters are systematically studied. Implementation of the isotope depletion techniques in conjunction with lower resoln. mass analyzers is discussed as well. The authors investigate theor. the position of the centroid of the isotopic mass distributions (centroid mass) and the shift between the monoisotopic and the centroid masses of biopolymers as a function of the isotope abundance (e.g., 12C:13C ratio). The behavior of other additive mass parameters, like the ratio between the monoisotopic and the first isotopic peak, is also discussed. We address by computer simulations the effects of different instrumental parameters like mass resoln. and ion statistics as a function of isotope abundances and from there the achievable mass accuracy for high-mass biopolymers. We assess some of the practical issues of the isotope depletion technique, viz., to what degree and with what accuracy the depletion procedure should be performed for achieving the desired mass accuracy.
    46. 46
      Gallagher, K. J.; Palasser, M.; Hughes, S.; Mackay, C. L.; Kilgour, D. P. A.; Clarke, D. J. Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins. J. Am. Soc. Mass Spectrom. 2020, 31 (3), 700– 710,  DOI: 10.1021/jasms.9b00119
      46
      Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins
      Gallagher, Kelly J.; Palasser, Michael; Hughes, Sam; Mackay, C. Logan; Kilgour, David P. A.; Clarke, David J.
      Journal of the American Society for Mass Spectrometry (2020), 31 (3), 700-710CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
      Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biol. MS expts., the practicality of applying top-down methodologies is still limited at higher mol. mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing no. of isotopic peaks as mol. mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago and has been demonstrated for several small proteins. Here the authors extend this approach, introducing a new highly efficient method for the prodn. of recombinant proteins depleted in 13C and 15N and demonstrating its advantages for top-down anal. of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal obsd. up to 50 kDa. In top-down fragmentation expts., the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, the authors demonstrate greatly increased sequence coverage in time-limited top-down expts.-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.
    47. 47
      Popovic, Z.; Anderson, L. C.; Zhang, X.; Butcher, D. S.; Blakney, G. T.; Zubarev, R. A.; Marshall, A. G. Analysis of Isotopically Depleted Proteins Derived from Escherichia Coli and Caenorhabditis Elegans Cell Lines by Liquid Chromatography 21 T Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2023, 34, 137– 144,  DOI: 10.1021/jasms.2c00242
      47
      Analysis of Isotopically Depleted Proteins Derived from Escherichia coli and Caenorhabditis elegans Cell Lines by Liquid Chromatography 21 T Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry
      Popovic, Zeljka; Anderson, Lissa C.; Zhang, Xuepei; Butcher, David S.; Blakney, Greg T.; Zubarev, Roman A.; Marshall, Alan G.
      Journal of the American Society for Mass Spectrometry (2023), 34 (2), 137-144CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
      Protein mass measurement by mass spectrometry is complicated by wide isotopic distributions that result from incorporation of heavy isotopes of C, H, N, O, and S, thereby limiting signal-to-noise ratio (SNR) and accurate intact mass detn., particularly for larger proteins []. Observation of the monoisotopic mass-to-charge ratio (m/z) is the simplest and most accurate way to det. intact protein mass, but as mass increases, the relative abundance of the monoisotopic peak becomes so low that it is often undetectable. Here, we used an isotopically depleted growth medium to culture bacterial cells (Escherichia coli), resulting in isotopically depleted proteins. Isotopically depleted proteins show increased sequence coverage, mass measurement accuracy, and increased S/N of the monoisotopic peak by Fourier transform ion cyclotron resonance mass spectrometry anal. We then grew Caenorhabditis elegans cells in a medium contg. living isotopically depleted E. coli cells, thereby producing the first isotopically depleted eukaryotic proteins. This is the first time isotopic depletion has been implemented for four isotopes (1H, 12C, 14N, and 16O), resulting in the highest degree of depletion ever used for protein anal. and further improving MS anal.
    48. 48
      Loginov, D. S.; Fiala, J.; Chmelik, J.; Brechlin, P.; Kruppa, G.; Novak, P. Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on TimsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis. ACS Omega 2021, 6 (15), 10352– 10361,  DOI: 10.1021/acsomega.1c00732
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      Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis
      Loginov, Dmitry S.; Fiala, Jan; Chmelik, Josef; Brechlin, Peter; Kruppa, Gary; Novak, Petr
      ACS Omega (2021), 6 (15), 10352-10361CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      Fast photochem. oxidn. of proteins (FPOP) is a recently developed technique for studying protein folding, conformations, interactions, etc. In this method, hydroxyl radicals, usually generated by KrF laser photolysis of H2O2, are used for irreversible labeling of solvent-exposed side chains of amino acids. Mapping of the oxidized residues to the protein's structure requires pinpointing of modifications using a bottom-up proteomic approach. In this work, a quadrupole time-of-flight (QTOF) mass spectrometer coupled with trapped ion mobility spectrometry (timsTOF Pro) was used for identification of oxidative modifications in a model protein. Multiple modifications on the same residues, including six modifications of histidine, were successfully resolved. Moreover, parallel accumulation-serial fragmentation (PASEF) technol. allows successful sequencing of even minor populations of modified peptides. The data obtained indicate a clear improvement of the quality of the FPOP anal. from the viewpoint of the no. of identified peptides bearing oxidative modifications and their precise localization. Data are available via ProteomeXchange with identifier PXD020509.
    49. 49
      Li, K. S.; Shi, L.; Gross, M. L. Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization. Acc. Chem. Res. 2018, 51 (3), 736– 744,  DOI: 10.1021/acs.accounts.7b00593
      49
      Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization
      Li, Ke Sherry; Shi, Liuqing; Gross, Michael L.
      Accounts of Chemical Research (2018), 51 (3), 736-744CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Assessment of protein structure and interaction is crucial for understanding protein structure/function relations. Compared to high-resoln. structural tools, including x-ray crystallog., NMR, and cryo-EM, and traditional low-resoln. methods, such as CD, UV-visible, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resoln. (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure. The methodol. relies on "painting" the reactive and solvent-exposed amino acid residues with chem. tags and using the pattern of modifications as footprints from anal. by bottom-up MS-based proteomics to deduce protein higher order structures. The outcome can refer to proteins in soln. or even in cells and is complementary to those of x-ray crystallog. and NMR. It is particularly useful in mapping protein-ligand interfaces and conformational changes resulting from ligand binding, mutation, and aggregation.Fast photochem. oxidn. of proteins (FPOP), in its original conception, is a type of hydroxyl-radical-based protein footprinting that uses a pulsed KrF laser (248 nm) to trigger hydrolysis of hydrogen peroxide to produce soln. hydroxyl radicals, which subsequently modify the protein in situ. The platform is expanding to adopt other reactive species including carbenes. The reactivity of the probe depends on the intrinsic reactivity of the radical with the residue side chain and the solvent accessibility of the residue as a function of the tertiary/quaternary structures. By introducing an appropriate scavenger to compete with hydroxyl radical self-quenching, the lifetime of the primary radicals is remarkably shortened to approx. microsecond. Thus, the sampling time scale of FPOP is much faster than hydrogen-deuterium exchange and other covalent labeling methods relying on nonradical reactions.The short footprinting time scale of FPOP offers two major advantages for protein structure elucidation: (1) it allows the protein to be interrogated in its native or near-native state with min. structural perturbation; (2) it exhibits high sensitivity toward alterations in protein higher order structures because its sampling time is short with respect to protein conformational changes and dynamic motion. In addn., the covalent and irreversible oxidn. by the hydroxyl radical provides more flexibility in the downstream proteomics workflow and MS anal., permitting high spatial resoln. with residue-specific information.Since its invention in 2005 by Hambly and Gross, FPOP has developed from proof-of-concept to a valuable biophys. tool for interrogating protein structure. In this Account, the authors summarize the principles and exptl. design of FPOP that enable its fast labeling and describe the current and unique capabilities of the technique in protein higher order structure elucidation. Application examples include characterization of amyloid β self-assembly, protein-ligand interactions with a special emphasis on epitope mapping for protein therapeutics (e.g., antibody, Fab, and adnectin), protein folding detailed to residue-specific folding kinetics, and protein flexibility/dynamics. Addnl., the utility of FPOP-based oxidative footprinting should grow with the authors' continuing developments of novel reagents (e.g., sulfate radical anion, carbene diradical, and trifluoromethyl radical). These reactive reagents are compatible with the current FPOP platform and offer different reactivity and selectivity toward various types of amino acid residues, providing complementary insights into protein higher order structures for sol. proteins and ultimately for membrane-bound proteins.
    50. 50
      Perez-Riverol, Y.; Bai, J.; Bandla, C.; García-Seisdedos, D.; Hewapathirana, S.; Kamatchinathan, S.; Kundu, D. J.; Prakash, A.; Frericks-Zipper, A.; Eisenacher, M.; Walzer, M.; Wang, S.; Brazma, A.; Vizcaíno, J. A. The PRIDE Database Resources in 2022: A Hub for Mass Spectrometry-Based Proteomics Evidences. Nucleic Acids Res. 2022, 50 (D1), D543– D552,  DOI: 10.1093/nar/gkab1038
      50
      PRIDE database resources in 2022 hub for mass spectrometry-based proteomics evidences
      Perez-Riverol, Yasset; Bai, Jingwen; Bandla, Chakradhar; Garcia-Seisdedos, David; Hewapathirana, Suresh; Kamatchinathan, Selvakumar; Kundu, Deepti J.; Prakash, Ananth; Frericks-Zipper, Anika; Eisenacher, Martin; Walzer, Mathias; Wang, Shengbo; Brazma, Alvis; Vizcaino, Juan Antonio
      Nucleic Acids Research (2022), 50 (D1), D543-D552CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      The PRoteomics IDEntifications (PRIDE) database is the world's largest data repository of mass spectrometry-based proteomics data. PRIDE is one of the founding members of the global ProteomeXchange (PX) consortium and an ELIXIR core data resource. In this manuscript, we summarize the developments in PRIDE resources and related tools since the previous update manuscript was published in Nucleic Acids Research in 2019. The no. of submitted datasets to PRIDE Archive (the archival component of PRIDE) has reached on av. around 500 datasets per mo during 2021. In addn. to continuous improvements in PRIDE Archive data pipelines and infrastructure, the PRIDE Spectra Archive has been developed to provide direct access to the submitted mass spectra using Universal Spectrum Identifiers. As a key point, the file format MAGE-TAB for proteomics has been developed to enable the improvement of sample metadata annotation. Addnl., the resource PRIDE Peptidome provides access to aggregated peptide/protein evidences across PRIDE Archive. Furthermore, we will describe how PRIDE has increased its efforts to reuse and disseminate high-quality proteomics data into other added-value resources such as UniProt, Ensembl and Expression Atlas.
    51. 51
      Boura, E.; Rezabkova, L.; Brynda, J.; Obsilova, V.; Obsil, T. Structure of the Human FOXO4-DBD–DNA Complex at 1.9 Å Resolution Reveals New Details of FOXO Binding to the DNA. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66 (12), 1351– 1357,  DOI: 10.1107/S0907444910042228
      51
      Structure of the human FOXO4-DBD-DNA complex at 1.9 Å resolution reveals new details of FOXO binding to the DNA
      Boura, Evzen; Rezabkova, Lenka; Brynda, Jiri; Obsilova, Veronika; Obsil, Tomas
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (12), 1351-1357CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      FOXO4 is a member of the FOXO subgroup of forkhead transcription factors that constitute key components of a conserved signaling pathway that connects growth and stress signals to transcriptional control. Here, the 1.9 Å resoln. crystal structure of the DNA-binding domain of human FOXO4 (FOXO4-DBD) bound to a 13 bp DNA duplex contg. a FOXO consensus binding sequence is reported. The structure shows a similar recognition of the core sequence as has been shown for two other FOXO proteins. Helix H3 is docked into the major groove and provides all of the base-specific contacts, while the N-terminus and wing W1 make addnl. contacts with the phosphate groups of DNA. In contrast to other FOXO-DBD-DNA structures, the loop between helixes H2 and H3 has a different conformation and participates in DNA binding. In addn., the structure of the FOXO4-DBD-DNA complex suggests that both direct water-DNA base contacts and the unique water-network interactions contribute to FOXO-DBD binding to the DNA in a sequence-specific manner.
    52. 52
      Flores, S. C.; Altman, R. B. Turning Limited Experimental Information into 3D Models of RNA. RNA 2010, 16 (9), 1769– 1778,  DOI: 10.1261/rna.2112110
      52
      Turning limited experimental information into 3D models of RNA
      Flores, Samuel Coulbourn; Altman, Russ B.
      RNA (2010), 16 (9), 1769-1778CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      Our understanding of RNA functions in the cell is evolving rapidly. As for proteins, the detailed three-dimensional (3D) structure of RNA is often key to understanding its function. Although crystallog. and NMR can det. the at. coordinates of some RNA structures, many 3D structures present tech. challenges that make these methods difficult to apply. The great flexibility of RNA, its charged backbone, dearth of sp. surface features, and propensity for kinetic traps all conspire with its long folding time, to challenge in silico methods for physics-based folding. On the other hand, base-pairing interactions (either in runs to form helixes or isolated tertiary contacts) and motifs are often available from relatively low-cost expts. or informatics analyses. We present RNABuilder, a novel code that uses internal coordinate mechanics to satisfy user-specified base pairing and steric forces under chem. constraints. The code recapitulates the topol. and characteristic L-shape of tRNA and obtains an accurate noncrystallog. structure of the Tetrahymena ribozyme P4/P6 domain. The algorithm scales nearly linearly with mol. size, opening the door to the modeling of significantly larger structures.
    53. 53
      Flores, S. C.; Bernauer, J.; Shin, S.; Zhou, R.; Huang, X. Multiscale Modeling of Macromolecular Biosystems. Brief. Bioinform. 2012, 13 (4), 395– 405,  DOI: 10.1093/bib/bbr077
      53
      Multiscale modeling of macromolecular biosystems
      Flores, Samuel C.; Bernauer, Julie; Shin, Seokmin; Zhou, Ruhong; Huang, Xuhui
      Briefings in Bioinformatics (2012), 13 (4), 395-405CODEN: BBIMFX; ISSN:1467-5463. (Oxford University Press)
      In this article, we review the recent progress in multiresoln. modeling of structure and dynamics of protein, RNA and their complexes. Many approaches using both physics-based and knowledge-based potentials have been developed at multiple granularities to model both protein and RNA. Coarse graining can be achieved not only in the length, but also in the time domain using discrete time and discrete state kinetic network models. Models with different resolns. can be combined either in a sequential or parallel fashion. Similarly, the modeling of assemblies is also often achieved using multiple granularities. The progress shows that a multiresoln. approach has considerable potential to continue extending the length and time scales of macromol. modeling.
    54. 54
      Černý, J.; Božíková, P.; Svoboda, J.; Schneider, B. A Unified Dinucleotide Alphabet Describing Both RNA and DNA Structures. Nucleic Acids Res. 2020, 48 (11), 6367– 6381,  DOI: 10.1093/nar/gkaa383
      54
      A unified dinucleotide alphabet describing both RNA and DNA structures
      Cerny, Jiri; Bozikova, Paulina; Svoboda, Jakub; Schneider, Bohdan
      Nucleic Acids Research (2020), 48 (11), 6367-6381CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      By analyzing almost 120 000 dinucleotides in over 2000 nonredundant nucleic acid crystal structures, we define 96+1 diNucleotide Conformers, NtCs, which describe the geometry of RNA and DNA dinucleotides. NtC classes are grouped into 15 codes of the structural alphabet CANA (Conformational Alphabet of Nucleic Acids) to simplify symbolic annotation of the prominent structural features of NAs and their intuitive graphical display. The search for nontrivial patterns of NtCs resulted in the identification of several types of RNA loops, some of them obsd. for the first time. Over 30% of the nearly six million dinucleotides in the PDB cannot be assigned to any NtC class but we demonstrate that up to a half of them can be re-refined with the help of proper refinement targets. A statistical anal. of the preferences of NtCs and CANA codes for the 16 dinucleotide sequences showed that neither the NtC class AA00, which forms the scaffold of RNA structures, nor BB00, the DNA most populated class, are sequence neutral but their distributions are significantly biased. The reported automated assignment of the NtC classes and CANA codes available at dnatco.org provides a powerful tool for unbiased anal. of nucleic acid structures by structural and mol. biologists.
    55. 55
      Černý, J.; Božíková, P.; Malý, M.; Tykač, M.; Biedermannová, L.; Schneider, B. Structural Alphabets for Conformational Analysis of Nucleic Acids Available at Dnatco.Datmos.Org. Acta Crystallogr. Sect. D Struct. Biol. 2020, 76 (9), 805– 813,  DOI: 10.1107/S2059798320009389
      55
      Structural alphabets for conformational analysis of nucleic acids available at dnatco.datmos.org
      Cerny, Jiri; Bozikova, Paulina; Maly, Michal; Tykac, Michal; Biedermannova, Lada; Schneider, Bohdan
      Acta Crystallographica, Section D: Structural Biology (2020), 76 (9), 805-813CODEN: ACSDAD; ISSN:2059-7983. (International Union of Crystallography)
      A detailed description of the dnatco.datmos.org web server implementing the universal structural alphabet of nucleic acids is presented. It is capable of processing any mmCIF- or PDB-formatted files contg. DNA or RNA mols.; these can either be uploaded by the user or supplied as the wwPDB or PDB-REDO structural database access code. The web server performs an assignment of the nucleic acid conformations and presents the results for the intuitive annotation, validation, modeling and refinement of nucleic acids.
    56. 56
      Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E. GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1–2, 19– 25,  DOI: 10.1016/j.softx.2015.06.001
      There is no corresponding record for this reference.
    57. 57
      Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from Ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
      57
      ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
      Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
      Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
    58. 58
      Liebl, K.; Zacharias, M. Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry. J. Chem. Theory Comput. 2021, 17 (11), 7096– 7105,  DOI: 10.1021/acs.jctc.1c00682
      58
      Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry
      Liebl, Korbinian; Zacharias, Martin
      Journal of Chemical Theory and Computation (2021), 17 (11), 7096-7105CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      An accurate mol. mechanics force field forms the basis of Mol. Dynamics simulations to obtain a realistic view of the structure and dynamics of biomols. such as DNA. Although frequently updated to improve agreement with available exptl. data, DNA force fields still rely in part on parameters introduced more than 20 years ago. We have developed an entirely new DNA force field, Tumuc1, derived from quantum mech. calcns. to obtain a consistent set of bonded parameters and partial at. charges. The performance of the force field was extensively tested on a variety of DNA mols. It excels in accuracy of B-DNA simulations but also performs very well on other types of DNA structures and structure formation processes such as hairpin folding, duplex formation, and dynamics of DNA-protein complexes. It can complement existing force fields in order to provide an increasingly accurate description of the structure and dynamics of DNA during simulation studies.
    59. 59
      Xu, G.; Chance, M. R. Radiolytic Modification and Reactivity of Amino Acid Residues Serving as Structural Probes for Protein Footprinting. Anal. Chem. 2005, 77 (14), 4549– 4555,  DOI: 10.1021/ac050299+
      59
      Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting
      Xu, Guozhong; Chance, Mark R.
      Analytical Chemistry (2005), 77 (14), 4549-4555CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examg. the structure and dynamics of biol. assemblies. In this study, the reactivities and tendencies to form easily detectible products for all 20 (common) amino acid side chains along with cystine are directly compared using various stds. Although we have previously reported on the oxidn. of many of these residues, this study includes a detailed examn. of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting expts. All 20 amino amides along with cystine and a few tripeptides were irradiated by γ-rays, the products were analyzed by electrospray mass spectrometry, and rate consts. of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal stds. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical expts. and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidn. products are not easily detectible. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical expts. Since these residues comprise ∼65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.
    60. 60
      Niu, B.; Gross, M. L. MS-Based Hydroxyl Radical Footprinting: Methodology and Application of Fast Photochemical Oxidation of Proteins (FPOP). In Mass Spectrometry-Based Chemical Proteomics; Wiley, 2019; pp 363– 416.  DOI: 10.1002/9781118970195.ch15 .
      There is no corresponding record for this reference.
    61. 61
      Yin, V.; Mian, S. H.; Konermann, L. Lysine Carbonylation Is a Previously Unrecognized Contributor to Peroxidase Activation of Cytochrome c by Chloramine-T. Chem. Sci. 2019, 10 (8), 2349– 2359,  DOI: 10.1039/C8SC03624A
      61
      Lysine carbonylation is a previously unrecognized contributor to peroxidase activation of cytochrome c by chloramine-T
      Yin, Victor; Mian, Safee H.; Konermann, Lars
      Chemical Science (2019), 10 (8), 2349-2359CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      The peroxidase activity of cytochrome c (cyt c) plays a key role during apoptosis. Peroxidase catalysis requires a vacant Fe coordination site, i.e., cyt c must undergo an activation process involving structural changes that rupture the native Met80-Fe contact. A common strategy for dissocg. this bond is the conversion of Met80 to sulfoxide (MetO). It is widely believed that this MetO formation in itself is sufficient for cyt c activation. This notion originates from studies on chloramine-T-treated cyt c (CT-cyt c) which represents a std.model for the peroxidase activated state. CT-cyt c is considered to be a "clean" species that has undergone selective MetO formation, without any other modifications. Using optical, chromatog., and mass spectrometry techniques, the current work demonstrates that CT-induced activation of cyt c is more complicated than previously thought. MetO formation alone results in only marginal peroxidase activity, because dissocn. of the Met80-Fe bond triggers alternative ligation scenarios where Lys residues interfere with access to the heme. We found that CT causes not only MetO formation, but also carbonylation of several Lys residues. Carbonylation is assocd. with -1 Da mass shifts that have gone undetected in the CT-cyt c literature. Proteoforms possessing both MetO and Lys carbonylation exhibit almost fourfold higher peroxidase activity than those with MetO alone. Carbonylation abrogates the capability of Lys to coordinate the heme, thereby freeing up the distal site as required for an active peroxidase. Previous studies on CT-cyt c may have inadvertently examd. carbonylated proteoforms, potentially misattributing effects of carbonylation to solely MetO formation.
    62. 62
      Liu, X. R.; Zhang, M. M.; Gross, M. L. Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications. Chemical Reviews 2020, 4355– 4454,  DOI: 10.1021/acs.chemrev.9b00815
      62
      Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications
      Liu, Xiaoran Roger; Zhang, Mengru Mira; Gross, Michael L.
      Chemical Reviews (Washington, DC, United States) (2020), 120 (10), 4355-4454CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Proteins adopt different higher-order structures (HOS) to enable their unique biol. functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS detn. of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS anal., through which different covalent labeling approaches "mark" the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen-deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resoln., the authors present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biol. questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a ref. for investigators seeking a MS-based tool to address structural questions in protein science.
    63. 63
      Boura, E.; Silhan, J.; Herman, P.; Vecer, J.; Sulc, M.; Teisinger, J.; Obsilova, V.; Obsil, T. Both the N-Terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 Are Important for DNA Binding. J. Biol. Chem. 2007, 282 (11), 8265– 8275,  DOI: 10.1074/jbc.M605682200
      63
      Both the N-terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 are Important for DNA Binding
      Boura, Evzen; Silhan, Jan; Herman, Petr; Vecer, Jaroslav; Sulc, Miroslav; Teisinger, Jan; Obsilova, Veronika; Obsil, Tomas
      Journal of Biological Chemistry (2007), 282 (11), 8265-8275CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      FoxO4 belongs to the "O" subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel β-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors. In this report, we investigated the role of the N-terminal loop (portion located upstream of first helix H1) and the C-terminal region (loop known as wing W2) of the forkhead domain of transcription factor FoxO4 in DNA binding. Although the deletion of either portion partly reduces the FoxO4-DBD binding to the DNA, the simultaneous deletion of both regions inhibits DNA binding significantly. Foerster resonance energy transfer measurements and mol. dynamics simulations suggest that both studied N- and C-terminal regions of FoxO4-DBD directly interact with DNA. In the presence of the N-terminal loop the protein kinase B-induced phosphorylation of wing W2 by itself has negligible effect on DNA binding. On the other hand, in the absence of this loop the phosphorylation of wing W2 significantly inhibits the FoxO4-DBD binding to the DNA. The binding of the 14-3-3 protein efficiently reduces DNA-binding potential of phosphorylated FoxO4-DBD regardless of the presence of the N-terminal loop. Our results show that both N- and C-terminal regions of forkhead domain are important for stability of the FoxO4-DBD·DNA complex.
    64. 64
      Obsilova, V.; Vecer, J.; Herman, P.; Pabianova, A.; Sulc, M.; Teisinger, J.; Boura, E.; Obsil, T. 14–3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4. Biochemistry 2005, 44 (34), 11608– 11617,  DOI: 10.1021/bi050618r
      64
      14-3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4
      Obsilova, Veronika; Vecer, Jaroslav; Herman, Petr; Pabianova, Anna; Sulc, Miroslav; Teisinger, Jan; Boura, Evzen; Obsil, Tomas
      Biochemistry (2005), 44 (34), 11608-11617CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The 14-3-3 proteins are a family of regulatory signaling mols. that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 Proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of its nuclear localization sequence (NLS). Masking or obscuring of the NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equil. of FoxO localization toward the cytoplasm. To the best of our knowledge, there is no exptl. evidence showing a direct interaction between the 14-3-3 protein and the NLS of FoxO. Therefore, the main goal of this work was to investigate whether phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of the FoxO4 NLS. We have used site-directed labeling of the FoxO4 NLS with the extrinsic fluorophore 1,5-IAEDANS in conjunction with steady-state and time-resolved fluorescence spectroscopy to study conformational changes of the FoxO4 NLS in vitro. Our data show that 14-3-3 protein binding significantly changes the environment around the AEDANS-labeled NLS and reduces its flexibility. On the other hand, phosphorylation itself and the binding of double-stranded DNA have a small effect on the structure of this region. Our results also suggest that the DNA-binding domain of FoxO4 remains relatively mobile while bound to the 14-3-3 protein.
    65. 65
      James, V. K.; Sanders, J. D.; Aizikov, K.; Fort, K. L.; Grinfeld, D.; Makarov, A.; Brodbelt, J. S. Advancing Orbitrap Measurements of Collision Cross Sections to Multiple Species for Broad Applications. Anal. Chem. 2022, 94 (45), 15613– 15620,  DOI: 10.1021/acs.analchem.2c02146
      65
      Advancing Orbitrap Measurements of Collision Cross Sections to Multiple Species for Broad Applications
      James, Virginia K.; Sanders, James D.; Aizikov, Konstantin; Fort, Kyle L.; Grinfeld, Dmitry; Makarov, Alexander; Brodbelt, Jennifer S.
      Analytical Chemistry (Washington, DC, United States) (2022), 94 (45), 15613-15620CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Measurement of collision cross section (CCS), a parameter reflecting an ion's size and shape, alongside high-resoln. mass anal. extends the depth of mol. anal. by providing structural information beyond mol. mass alone. Although these measurements are most commonly undertaken using a dedicated ion mobility cell coupled to a mass spectrometer, alternative methods have emerged to ext. CCSs directly by anal. of the decay rates of either time-domain transient signals or the FWHM of frequency domain peaks in FT mass analyzers. This information is also accessible from FTMS mass spectra obtained in commonly used workflows directly without the explicit access to transient or complex Fourier spectra. Previously, these expts. required isolation of individual charge states of ions prior to CCS anal., limiting throughput. Here we advance Orbitrap CCS measurements to more users and applications by detg. CCSs from commonly available mass spectra files as well as estg. CCS for multiple charge states simultaneously and showcase these methods by the measurement of CCSs of fragment ions produced from collisional activation of proteins.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c03759.

    • Additional methods: Expression and purification of isotopically natural and depleted FOXO4-DBD, top-down data processing, electrophoretic mobility shift assay; Additional Figures: (Figure S1) FOXO4 sequence with both top-down and wild-type numbering, (Figure S2) ESI-MS spectra of desalted protein samples, (Figure S3) EMSA gel, (Figure S4) broadband ECD spectrum of IN-/ID-FOXO4-DBD, (Figure S5) quantified ions of IN-FOXO4-DBD, (Figure S6) zoom of [c4]1+ and [c5]1+ ions, (Figure S7) zoom of [z3]1+ and [z4]1+ ions, (Figure S8) quantified ions of ID-FOXO4-DBD, (Figure S9) zoom-in of ECD MSMS spectra of IN-/ID-FOXO4-DBD, (Figure S10) ab initio model of FOXO4-IRE with wild-type numbering, (Figure S11) bottom-up analysis of IN-FOXO4-DBD, (Figure S12) quantified extent of oxidation of W94 residue; Additional table: (Table S1) all modifications identified in bottom-up approach (PDF)


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