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Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS

  • Andrea F. G. Gargano*
    Andrea F. G. Gargano
    Center for Analytical Sciences Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands
    Vrije Universiteit Amsterdam, Department of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, de Boelelaan 1085, 1081HV Amsterdam, The Netherlands
    *E-mail: [email protected]. Tel: +31-20-525 7040.
    More by Andrea F. G. Gargano
    Orcidhttp://orcid.org/0000-0003-3361-7341
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  • Jared B. Shaw
    Jared B. Shaw
    Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
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    Orcidhttp://orcid.org/0000-0002-1130-1728
  • , 
  • Mowei Zhou
    Mowei Zhou
    Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
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    Orcidhttp://orcid.org/0000-0003-3575-3224
  • , 
  • Christopher S. Wilkins
    Christopher S. Wilkins
    Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
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  • Thomas L. Fillmore
    Thomas L. Fillmore
    Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
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  • Ronald J. Moore
    Ronald J. Moore
    Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
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  • , 
  • Govert W. Somsen
    Govert W. Somsen
    Center for Analytical Sciences Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands
    Vrije Universiteit Amsterdam, Department of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, de Boelelaan 1085, 1081HV Amsterdam, The Netherlands
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  • , and 
  • Ljiljana Paša-Tolić
    Ljiljana Paša-Tolić
    Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
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Cite this: J. Proteome Res. 2018, 17, 11, 3791–3800
Publication Date (Web):September 18, 2018
https://doi.org/10.1021/acs.jproteome.8b00458

Copyright © 2022 American Chemical Society. This publication is licensed under CC-BY-NC-ND.

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Abstract

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Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 μg).

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Introduction

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Histones are highly heterogeneous chromatin proteins with an isoelectric point of ∼11 and a molecular mass of 10–22 kDa. The histone octamer is composed of two H2A, H2B, H3, and H4 proteins assembled as one H3–H4 heterotetramer and two H2A-H2B heterodimers, with roughly 147 bp of DNA wrapped around it. (1) This protein–DNA complex is the basic structural unit of chromatin, the nucleosome. Post-translational modifications (PTMs) of histones have defined roles in regulating gene transcription. (2−4) For instance, increased acetylation is typically associated with gene activation due to a more open chromatin structure that makes it accessible to transcription factors. Another example is methylation, which can be associated with either transcriptional repression or activation, depending on the site and degree of methylation. (1−3)
Traditionally, histone samples are studied using antibody-based enrichment methods. (5) However, immunoprecipitation methods are unable to identify novel PTMs, as they require previous knowledge of the type of modification and a highly specific antibody for each modification studied. Moreover, many antibodies are not entirely site specific and can cross-react with similar modifications on different residues. (6)
Mass spectrometry (MS) based analyses of histone PTMs have emerged as an attractive alternative to traditional antibody-based methods. (7−12) Bottom-up methods, employing protein digestion prior to liquid chromatography (LC)-MS analysis, are established approaches to characterize proteins and their PTMs. However, a limitation of these strategies is their inability to assign a specific modified peptide sequence to a particular proteoform. (13) Histones, in particular, are rich in lysine and arginine, and bottom-up methods using trypsin cleave histones into peptides that are too short for effective LC-MS analysis. Additionally, connectivity between peptides is lost making it impossible to retrieve exact proteoforms. (10,14) Middle-down methods performed using enzymes with rare cleavage sites or chemically blocking unmodified lysine can generate large peptides (about 30–60 amino acid residues). Analysis of longer peptides enables identification of combinatorial PTMs and the crosstalk between PTMs. (10,15,16) However, characterization of proteins like histones H2B requires analysis of the intact protein due to variants that differ by one or a few amino acids near both the N- and C-termini. Complete characterization of the combinatorial distribution of histone proteoforms is only possible via the analysis of intact histones using top-down proteomic approaches. (17−19) Quantitative analysis of proteoforms of isolated histone H4 (20) and H2A and H2B (21) have been recently reported demonstrating the applicability of such workflows to biological studies. However, despite recent improvements in instrumentation and methodology, top-down analyses still face many practical challenges in sample preparation, separations, MS detection, and informatics. (22)
For histones, isolation of nuclei from other cellular contents is usually preferred to obtain sufficiently pure histones for in-depth PTM profiling. However, the yield of nuclei isolation and histone extraction can vary significantly across organisms and cell types, limiting the amount of histone proteins available for LC-MS analysis. Even for global top-down analyses, sample prefractionation is typically needed to enable comprehensive coverage of the various proteoforms. Therefore, advanced LC methods that offer a streamlined purification and fractionation process would be highly beneficial.
The two most commonly used separation methods in the top-down analysis of histones are weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) and reversed phase liquid chromatography (RPLC). The ion exchange nature of WCX-HILIC makes this separation method selective toward histone charge variants (e.g., resulting from lysine acetylation). (23) However, WCX-HILIC has low MS compatibility and limited separation efficiency. RPLC provides good MS compatibility but has limited proteoform selectivity and primarily resolves the main histone families (H1, H2A, H2B, H3, and H4). (24)
Here we present an online nanoflow LC×LC method that enables comprehensive characterization of histone proteoforms. The 2DLC setup couples a WCX-HILIC using a shallow gradient in the first dimension to a fast RPLC in the second separation dimension. The two liquid phase separations are coupled using a modulation interface having trap columns (active modulation, a×m) allowing both separations to run at a flow rate of 300 nL/min. Our separation setup was coupled to a Q Exactive HF Orbitrap mass spectrometer modified to incorporate 193 nm ultraviolet photodissociation (UVPD). (25−27)
By combining the WCX-HILIC/a×m/RPLC with UVPD-HRMS, we were able to identify hundreds of HeLa core histone proteoforms using a small amount of starting material (∼1.5 μg). These results suggest that 2DLC-MS platforms could significantly improve our ability to analyze proteoforms present in biologically relevant samples available in limited amounts (e.g., derived from laser microdissection).

Experimental Section

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Chemicals and Reagents

Formic acid (FA), ammonium formate, and ammonium acetate were purchased from Sigma-Aldrich. The solvents used were Milli-Q grade water (18.2 mΩ) and mass spectrometry grade acetonitrile (ACN). HeLa core histones were purchased from ActiveMotif (Carlsbad, CA). The sample was buffer exchanged into water using 3 kDa molecular weight cutoff filters (EMD Millipore, Billerica, MA) and stored at −70 °C. We prepared a histone sample for WCX-HILIC and HILIC/a×m/RPLC analysis as a 0.52 μg/μL solution in 50/50 ACN/5 mM ammonium acetate (v/v). Samples for RPLC were prepared in the same concentration but dissolved in 2% ACN 0.1%FA. All the columns and trap columns used in this study were packed in-house.

1DLC Analysis (WCX-HILIC and RPLC Analysis)

One dimensional LC analyses (1DLC) were done using a Waters M-Class LC operated using MassLynx. The WCX-HILIC separation method was developed based on workflow described by Tian et al. (28) The chromatographic column used was a silica based weak cation exchanger (PolyCAT A, 5 μm particles, 1000 Å pore size; Poly LC, Columbia, MD, USA) packed in a 150 mm × 75 μm I.D. column. The optimized method used 70/29/1 ACN/water/FA as mobile phase A and 65/27/8 ACN/water/FA (v/v/v) as mobile phase B. The analyses were done at 300 nL/min using a gradient from 0% B to 100% B in 100 min, 20 min at 100% B, and with 30 min of equilibration at 0% after each injection. Injections were done using a 5 μL loop in partial injection mode (solvent, 90/10 ACN/10 mM ammonium acetate).
We performed the 1D RPLC separation on a 700 mm long 75 μm ID column packed with C18 silica (Jupiter C18, 3 μm particles, 300 Å pore size, Phenomenex, Torrance, CA, USA). We used 0.1% FA in water as mobile phase A and 0.1% FA in ACN as B. The sample was injected using a 20 μL loop in full loop mode and trapped on a C18 trap column (Phenomenex Aeris XB-C18, 3.6 μm particles, 200 Å pore size, 50 mm × 100 μm). The sample was desalted on the trap cartridge for 20 min at 5% solvent B at a flow rate of 2.5 μL/min. Subsequently, the flow was reduced to 0.3 μL/min and directed to the C18 column. For elution we used a linear gradient starting at 10% solvent B and increasing to 20% B in 5 min, and ramped to 50% B at 100 min then to 80% B in 10 min and washing the column for 20 min at 80% B and then re-equilibrated at 5% B for 20 min.

Online Comprehensive Two-Dimensional LC (WCX-HILIC/a×m/RPLC, 2DLC)

The two-dimensional separation platform was assembled from Waters M-Class LC modules (2 gradient-pumps, one used as gradient pump for the 2D column and one for delivering the dilution flow; autosampler with 5 μL loop installed), one Acquity NanoLC pump (gradient pump used for the 1D column), and a nanoswitching valve from VICI Valco (PN C72MX-6670D 10-ports two-positions). We chose to use this valve because of the reduced port to port valve volume (approximately 30 nL). A schematic diagram of the 2DLC system is shown in Figure 2. We used two computers running MassLynx to control the online 2DLC system, one unit (PC1) controlling the first-dimension pump (1D) and one (PC2) controlling the autosampler (connected to the 1D column, WCX-HILIC), second dimension pump (2D), and the switching valve. The two LC dimensions and MS were synchronized using contact closure relays.
For the first LC separation, we used the same column described for the WCX-HILIC separation. The second-dimension column was packed in a 100 mm × 50 μm ID column, using reversed phase material (Jupiter C18, 3 μm particles, 300 Å pore size, Phenomenex, Torrance, CA, USA). Trap columns were prepared in a 50 mm × 100 μm ID columns using C18 core–shell particles (Aeris C18, 3.6 μm particles, 200 Å pore size, Phenomenex, Torrance, CA, USA).
We used mobile phase A1 70/29/1 ACN/water/FA and mobile phase B1 65/27/8 ACN/water/FA (v/v/v) for 1D. 2D used mobile phase A2 (0.1% FA in water) and B2 (0.1% FA in ACN). We applied gradient elution in both dimensions.
A constant flow of 1.6 μL/min of 1% ACN 0.1% TFA (dilution flow) was pumped via a tee union (VICI-Valco, C360QTPK2) connecting to the WCX-HILIC column and the modulation interface.
To complete the elution in the 1D and reduce carry over between analyses, we programmed the autosampler to perform full loop injection (5 μL plugs) from vials containing mobile phases with increasing water content (the solvent compositions are reported below) at the end of the 1D gradient.
PC2 triggered both the LC and MS run via the second pump module (2D) and was responsible for the injection on the WCX-HILIC column (1D). The 2D separations were programmed as a sequence of runs. We programmed the sequence of 2D separations with the first method injecting the sample, followed by 13 injections where no sample nor solvent was injected (“blank” injection), 5 analyses (15th, 16th, 17th, 18th and 19th  2D) where 5 μL of, respectively, 65/8/27, 60/8/32, 50/8/42, 25/8/67, 5/8/87 ACN/FA/water were injected, and a last 2D method (20th) where no sample nor solvent was injected. The total analysis time per run was 275 min.

Mass Spectrometry and Ultraviolet Photodissociation

Both the 1DLC configurations (WCX-HILIC and RPLC) as well as the WCX-HILIC/a×m/RPLC setup were coupled via a nanoelectrospray (ESI) source to a Q Exactive HF mass spectrometer (ThermoFisher Scientific Inc.) modified in a similar fashion as previously described to enable UVPD in the HCD cell. (29) The ESI voltage was set at 1.6 kV, with the inlet capillary temperature at 270 °C and S-RF lens at 70%. All the experiments were performed at a nominal resolving power of 120 000 (m/z = 200) for MS1 and 60 000 for MS/MS experiments. MS and MS/MS automatic gain control (AGC) target values were set at 3e6 (50 ms as maximum injection time) and 5e5 (200 ms), respectively. The number of microscans for MS1 and MS/MS was 3 and 6, respectively. The MS data were acquired as profile data, for 500 < m/z < 2000. Data dependent top 5 acquisitions (AGC minimum 2e4, intensity threshold 1e5) were performed with a 2 Th isolation window followed by UVPD using a single 1 mJ pulse. Dynamic exclusion was implemented with an exclusion duration of 60 s. MS/MS was only performed on species with charge states greater than four.

Data Analysis

Data analysis was performed using ProSightPC 4.0 (ThermoFisher Inc.) and Informed Proteomics (https://github.com/PNNL-Comp-Mass-Spec/Informed-Proteomics). (30) Prosight PC 4.0 was used to generate the results reported in Tables 1 and 2, and Informed Proteomics was used as a visualization and confirmatory tool to annotate the IDs (examples of protein identifications are collected in Supporting Materials S6 and S7).
Table 1. Histone Proteoforms Identified by the Injection of 1.5 μg of HeLa Core Histones into the RPLC, WCX-HILIC, or WCX-HILIC/axm/RPLC Coupled with UVPD-HRMSa
LC-MS methodH2AH2BH3H4Total unique ID
1DLC RPLC–UVPD-MSb19 (42)12 (23)36 (59)16 (39)83 (163)
1DLC WCX-HILIC–UVPD-MSb6 (7)10 (10)173 (177)19 (21)208 (215)
2DLC WCX-HILIC/a×m/RPLC-UVPD-MS R1c26 (29)12 (14)299 (299)29 (37)366 (379)
2DLC WCX-HILIC/a×m/RPLC-UVPD-MS R2c33 (39)14 (16)293 (298)21 (30)361 (383)
2DLC WCX-HILIC/a×m/RPLC-UVPD-MS R3c30 (33)13 (16)327 (338)21 (25)391 (412)
a

The identifications are obtained from searches against histone and contaminants database using Prosight PC (see Experimental Section for details). The data in parentheses indicate the total number of identifications, including the one from truncated forms (absolute and biomarker data search). Data filtered for P < 1E–4. Protein IDs available in Table S3 of the Supporting Information.

b

150 min acquisition time.

c

270 min acquisition time.

Table 2. Unique Histone Proteoforms Identified Using ProSight Absolute Mass Search against the Human Proteomea
LC-MS methodC < 33 ≤ C ≤ 45C > 45Total Unique IDs with C > 3 (total IDs)
1DLC RPLC–UVPD-MS147511667 (155)
1DLC WCX-HILIC–UVPD-MS10829837 (128)
2DLC WCX-HILIC/a×m/RPLC-UVPD-MS R1158612887 (173)
2DLC WCX-HILIC/a×m/RPLC-UVPD-MS R2175623092 (189)
2DLCWCX-HILIC/a×m/RPLC-UVPD-MS R3173613192 (190)
a

Identifications are sorted according to their C score. The total number of identified proteoforms is given in parentheses. Data filtered for P < 1E–4. Protein IDs available in Table S4 of the Supporting Information.

Two search modes of ProSight PC were used: absolute mass search to identify full-length proteins and biomarker search to identify protein fragments. Absolute mass search restricts the search space to proteins matching the mass of the precursor, whereas, in biomarker search, the search space is extended to protein sequence fragments matching the mass of the precursor. The data analysis was about 2 days per data set when performing absolute mass and biomarker searches in Prosight PC.
Alternatively, we created a database containing all histone proteins and common protein contaminants found in our samples (62 proteins). The restricted protein list was created from searches using Prosight PC performing absolute mass searches against the complete Human proteome using 1D (RPLC and WCX) and 2DLC-HRMS data allowing for 5 PTMs and 10 ppm mass accuracy for matching parent ions and fragment ions (reported in Table S2 of the Supporting Information). The database was created allowing for a maximum of 13 PTMs per protein.
Table 1 lists proteins identified using Prosight PC 4.0 for searches against the confined database and applying UVPD fragmentation settings for top-down MS2. RAW files were processed using the Xtract algorithm and Top Down (MS2) default settings. The analysis was performed in Absolute mass mode with a mass tolerance of 10 ppm for precursor and 10 ppm for fragments using the Δm mode (this function allows identification of proteoforms with PTMs not included in the annotated proteoform database). The unmatched spectra were further searched in Biomarker discovery mode, using the same mass tolerance settings. Histone identifications were filtered using the best hit per experiment function and a P < 1 × 10–04 cutoff. Analysis of a single data set with this approach took 2 days on a personal computer with an Intel Core i7-3820 CPU processor at 3.6 GHz with 32 GB of RAM. Additionally, we searched all data sets against the “histone&contaminants database” using the Informed-Proteomics workflow: (30) ProMex software was used for intact mass deconvolution, MSPath-Finder was used as the search engine, and LcMsSpectator as data visualization tool. The feature maps reported in various figures were obtained using LcMsSpectator. The deconvolution and search settings were similar to the one in Prosight PC. MSPath-Finder does not use previous knowledge regarding the PTM site and type of modification (e.g., from uniprot.org). Our searches allowed for a maximum of 5 PTMs per protein and mono-, di-, and trimethylation, acetylation, and phosphorylation.
The results reported in Table 2 were generated from ProsightPC using absolute mass searches against the complete human proteome, with a mass tolerance of 10 ppm for the precursor and 10 ppm for fragments, allowing up to 13 PTMs (but removing the Δm search mode) and filtered using the P < 1 × 10–04 cutoff and according to C score results. (31)
The mass spectrometry data have been deposited to the PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the data set identifier PXD008978.

Results and Discussion

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Top Down Analysis of Histones by 1DLC-MS

Weak cation exchange LC (WCX) separations using poly(aspartic acid) functionalized silica materials and mobile phases rich in ACN (e.g., 70% ACN) have been used to study the distribution of intact (23,28,32,33) and “partially digested” (middle down) proteoforms of histones. (16,34−36) The combination of ion exchange and hydrophilic interactions makes these separations selective toward PTMs that modify the charge of the protein (e.g., lysine acetylation). However, in order to ensure the complete elution of these basic proteins, typical separation methods are performed using high acid (e.g., 8% to 10% FA) or salt (e.g., 1 M NaClO4) concentration, hindering in some cases the direct coupling of WCX-HILIC with MS. Coupling to MS is possible using volatile additives. However, their high concentration can cause ion suppression and/or ESI instability, requiring frequent cleaning of the ion source, thus making the WCX-HILIC technique nonideal for high-throughput analyses.
Despite these drawbacks, WCX-HILIC offers excellent resolving power for acetylated and methylated species. (32,33,37)Figure 1a shows total ion chromatogram and deconvoluted mass vs retention time (i.e., LC-MS feature map) reconstructed from the analysis of 1.5 μg of HeLa histones. In our experiments, gradients similar to the one described by Garcia et al. (16) (using 0.5% FA and 20% of ACN) were not entirely successful in eluting proteoforms of H3 histones with low amounts of acetylation and methylation. We therefore applied the strategy reported by Tian et al. (28) where a high concentration of formic acid enables more efficient elution of H3 histones.

Figure 1

Figure 1. TIC LC-MS chromatogram (top) and feature map (deconvoluted mass spectra vs time) LC-MS chromatograms of 1DLC analysis (a) WCX-HILIC run and (b) RPLC MS. The colored areas indicate the elution zone of the different histone groups.

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The WCX-HILIC separation under the conditions described provides a continuous elution of histone proteins, where proteoforms with a higher degree of acetylation (thus higher mass) elute first, followed by the less modified proteoforms. Previous studies have described the retention mechanism for this separation, suggesting the acetylation of arginine or lysine residues as the main driving force for chemical discrimination. (28,35,38) Acetylation decreases the basicity of histones and thus weakens the electrostatic interaction with the stationary phase (polyaspartic acid). However, as can be seen in Figure 1a almost no discrimination between histone families is possible using this separation approach. The LC-MS feature maps for each histone group are reported in Supporting Material S3.
Top-down MS studies of histones using RPLC are typically done employing water to ACN gradients in the presence of an acidic modifier and can be easily coupled to MS via ESI. Different types of stationary phases have been used to separate histone families (H1, H2, H3, and H4), including C4, C5, C8, (23,33,39−41) and C18 materials. (7,42,43)Figure 1b shows the LC–MS feature map obtained from the injection of 1.5 μg of HeLa core histones on a C18 RPLC column. With this separation, the major histone families are resolved into different regions based on their hydrophobicity (defined primarily by amino acid composition and mass). H4 and H2B elute early, followed by H2A, and finally H3 proteoforms elute over a broad retention time range.
WCX-HILIC and RPLC methods target different sample properties (44) (i.e., charge vs hydrophobicity), and their coupling in various arrangements has proven to increase the overall number of identified species relative to one-dimensional methods. (23,28,45) Although more informative, these analytical workflows are not common due to potential sample losses (inherent to offline fractionation) and long analysis time. Initial research using offline 2DLC workflows used up to 100–150 μg of purified histones proteins. (45) Miniaturized fractionation columns reduced the quantity of sample needed for offline configurations to 7.5 μg. (24) Although the sample consumption can be reduced by fractionating the sample using smaller column IDs, this does not help to reduce the overall analysis time. For instance, Tian et al. (28) described an offline 2DLC workflow for fractionation of 7.5 μg of purified histone by capillary RPLC followed by sequential WCX-HILIC analyses. Despite reducing the sample quantity, the overall analysis time was over 900 min. Such a long analysis time limits the practicality of this workflow for high-throughput applications
The only online 2DLC setup for the analysis of histone proteoforms described so far is the multiple heart-cut system reported by Tian et al. (23) In this setup, multiple loops were used to collect 20 fractions from a 300 min WCX-HILIC separation using nine switching valves, several trap columns, and two RPLC columns. This configuration demonstrated a significant increase in the number histone proteoform identifications but required a large sample quantity (24 μg) and long analysis time (more than 60 h for a single analysis) which significantly diminished its applicability. This inspired us to develop a nanoflow online 2DLC method that would combine the advantages provided by coupling independent separation modalities with a small sample requirement and shorter analysis time.
In comparison to the online 2DLC platform previously described, our system has several advantages including the use of a single switching valve (two including the injector) and two columns running at low flow rates (300 nL/min). Switching from capillary to nanoLC regimes is nontrivial because in order to minimize the dead times between each 2D separation the design of the connections and synchronization of the pump hardware need to be carefully optimized. Handling smaller volumes results in less dilution thus allowing for the reduction of the sample size from the 24 μg reported in ref (23) to 1.5 μg (roughly 20×). Moreover, the first separation (WCX-HILIC) runs throughout the entire analysis using a long and shallow gradient that allows for high resolution fractionation. The system is programmed so that all the second dimension gradients are performed by the time the first dimension is completed. The setup described by Tian et al. (23) collects 20 fractions from a 300 min WCX-HILIC separation, and each is analyzed using a 180 min RPLC gradient for a total of >60 h per analysis, while here each fraction was analyzed using a 13 min RPLC analysis resulting in 275 min per 2DLC analysis.
Additionally, we performed UVPD fragmentation to increase the confidence in histone proteoform identification. (19)

Online Nanoflow LC×LC Using Active Modulation (2DLC)

In order to effectively couple two capillary based separations, it is important to minimize dead volumes in both LC dimensions and to ensure that the injection volume to the second dimension is sufficiently small to minimize cycle times and peak broadening.
For this reason, we substituted empty loops, typically used in LC×LC modulation interfaces (“passive modulation”), by capillary trap columns (“active modulation”). We previously described the principle of this approach in analytical (46) and low-flow (47) applications. These studies demonstrate how active modulation preserves the high LC separation power of online LC×LC and reduces sample dilution.
The nanoflow LC×LC setup combines a 150 mm × 75 μm ID WCX column separation at 200 nL/min, using a formic acid gradient from 70% ACN 1% FA to 68% ACN 8% FA, with a 100 mm × 50 μm ID C18 RPLC column separation at 300 nL/min using a water 0.1% FA to ACN 0.1% FA gradient. The ACN-rich eluent from the first dimension (strong eluting solvent in RPLC) is diluted 1:10 with water (1.6 μL/min) using a T-piece mixer. The eluent is loaded on 50 mm × 100 μm ID RPLC trap columns installed on a 10-port two-position nano HPLC valve. While a fraction eluting from the 1D is concentrated on a first trap, the second trap is used to inject a previously collected fraction onto the second-dimension column where a fast (10 min gradient time) separation takes place. The repetition of this process enables the sampling of several 1D fractions that are then simultaneously analyzed on the 2D. Using a low-dead volume switching valve (port to port volumes of 30 nL) and custom-made traps (with a mobile phase volume of about 250 nL) we were able to reduce the dead volume of the modulation unit to about 300 nL and complete 2D separations within a cycle time of 13 min (300 nL/min).
After the 1D gradient reached 100% B, we used the sampler injector to deliver solutions containing 8% FA and an increasing % of water to allow for elution of the most hydrophilic species retained on the 1D column, and thus ensure complete elution of proteins and other contaminants off the WCX column. Subsequently, we equilibrated the 1D column at a higher flow rate to prepare the column for the next injection. A schematic representation of the valve setup and gradient programming is depicted in Figure 2. Such an online nano 2DLC system represents the first of this kind, as the coupling of two nanoscale separation is a result of careful optimization of dead volumes, gradient delivery times, connection, and valve assembly to remove sources of air bubbles.

Figure 2

Figure 2. (a) Schematic representation of the 2DLC-MS/MS setup (WCX-HILIC/a×m/RPLC UVPD-HRMS). The sample components are separated using a gradient from 70% ACN 1%FA (A1) to 68% ACN 8% FA (B1; more details about the gradient programming are reported in the Experimental Section) on a WCX column, fractionated (online) using two trap columns (C18) and sequentially separated using a RPLC column (C18) with a water (A1) to ACN gradient (A2). During the analysis a dilution flow is delivered to reduce the elution strength of the mobile phase of the 1D (dilution flow rate ≈ 9× 1D flow rate) and allow the concentration of the analytes on a trap column (T1). Once the valve is switched, the analytes are eluted from the trap and separated on the 2D column. (b) Diagram reporting the gradient programmed for the 1D (WCX-HILIC) and 2D (RPLC); the green lines mark the injection events on the 1D column, while the blu and red trace the 1D and 2D gradient, respectively.

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The nanoflow LC×LC setup eliminates sample losses typically associated with offline fractionation. Moreover, the nanoflow rates and small ID (50 μm) of the 2D column limits sample dilution during analysis. This allows injection of the same quantity of protein as used in our RPLC and WCX-HILIC experiments (1.5 μg) without degrading sensitivity.
Figure 3a shows the 2DLC chromatogram of HeLa core histones aligned according to the 2D cycle time. Histone charge variants are separated by WCX-HILIC while RPLC separates histone families (H4 and H2B elute at the beginning of each RPLC analysis, followed by H2A and H3; the TIC of a single RPLC 2D run is reported in Figure S5 of the Supporting Information). Figure 3b overlays the TIC and LC-MS feature map, indicating the presence of the three histone families in most of the (2D) RPLC separations.

Figure 3

Figure 3. WCX-HILIC/a×m/RPLC UVPD-HRMS analysis of HeLa core histones. (a) Folded 2D chromatogram extracted from the total ion chromatograms (TICs). 2DLC separation combines the charge resolving power from the WCX separation with the histone family group separation (H2, H3, H4) afforded by RPLC. (b) TICs (top) of the 2DLC separation and the respective neutral mass spectra (bottom).

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Figure 4 shows H3 and H4 histone regions of the LC-MS feature map obtained with WCX-HILIC/a×m/RPLC. The distribution of masses over the WCX-HILIC retention window indicates that highly modified species elute early, following the separation trend that was observed when using WCX-HILIC alone. MS/MS analysis revealed that 1D histone proteoform separation is maintained and is mostly driven by the degree of acetylation; 2D further separates 1D fractions into different histone families.

Figure 4

Figure 4. Detailed view extracted from Figure 3 showing the neutral mass region of H3 (a) and H4 (b) histones. Species with a higher degree of acetylation (Ac) (i.e., with higher molecular mass) have lower retention in WCX-HILIC. Information on histone identifications is provided in Supporting Material S6.

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Online WCX-HILIC/a×m/RPLC offers the advantage of coupling WCX to MS detection using RPLC mobile phases, allowing to preservative the separation selectivity of this technique but removing the high acid concentration used for elution in WCX (source of ion suppression). In addition, RPLC resolves histone proteoform fractions into families and compresses the (wide) peaks from WCX-HILIC (up to 10 min in some cases) to widths of less than 2 min for each RPLC analysis. This, in combination with the use of a 50-μm ID 2D column, increases the sensitivity of the technique, allowing detection of lower abundance proteoforms that would not be detected in a 1D WCX-HILIC MS experiment.
Triplicate analyses of HeLa core histones showed similar elution profiles as shown Supporting Material S5. The major source of variation appears to be the fractionation of the WCX-HILIC analysis in the first dimension in combination with potential degradation of the sample during the analysis process, which results in shifts in the concentrations present in the RPLC fraction.
To further compare the results obtained from one- and two-dimensional separations, we extracted deconvoluted mass features corresponding to the H2A/B, H3, and H4 proteoforms (11200 < Mr < 11500 for H4, 13650 < Mr < 14150 for H2A/B, 15100 < Mr < 15500 for H3) and then combined their masses into 1 Da bins (the results are plotted in Figure S6 of the Supporting Information, and the corresponding feature maps are in Supporting Material S3). This visualization of our data set removes the discrimination coming from the retention time and thus the distinction of isobaric compounds but represents a simple method to compare the MS1 features detected. From this comparison we observed that more MS features not detected by 1D WCX or RPLC alone are observed in 2DLC experiments, especially for H3 histones (H4:66 nLC×LC, 54 WCX-HILIC, 65 RPLC; H2A/B 125 nLC×LC, 59 WCX, 99 RPLC and H3:160 nLC×LC, 67 WCX, 62 RPLC). It is likely that part of the MS features not detected either by WCX or RPLC are due to proteoform coelution and consequent ion suppression.

Proteoform Identification

The workflow used in this study included high resolution MS1 (120 000 at m/z = 200) followed by isolation of the five most abundant precursor ions for subsequent UVPD fragmentation (an illustration of the identification process is reported in Figure S1 of the Supporting Information). Similar to the approach described by Tian et al., (28,42) we initially searched MS/MS data against a database containing histones and major protein contaminants (the complete list is reported in Table S2 of the Supporting Information). In Table 2 we report the results for the 1D RPLC, 1D WCX-HILIC and LC×LC WCX-HILIC/a×m/RPLC analyses using Prosight PC.
A direct comparison of the identification results published using other offline and online 2DLC approaches (Tian (28,42)) is not possible, as the data were produced using different fragmentation approaches on a different mass spectrometer and data processing. We refer to Greer et al. (19) for a comparison of the results of online RPLC with UVPD fragmentation with respect to HCD and EThcD.
The number of identifications reported (1D and 2DLC) may appear lower relative to what has been reported for other histone top-down studies. (28) This is likely due to the tighter tolerances for precursor and fragment ions (10 ppm vs 1 to 10 Da reported previously (8,28)). Analyses using relaxed parameters to increase the number of proteoforms identified, but we preferred using restrictive matching settings to increase the confidence in the data presented.
Using WCX and WCX-HILIC/a×m/RPLC we predominantly identified intact proteoforms while RPLC predominantly identified truncated proteoforms. The shallow gradient and long column used in RPLC-MS allow the truncated forms (lower in abundance) to be resolved from their intact proteins and elute throughout the analysis. In contrast, in WCX-HILIC the truncated forms elute only in the initial part of the run and are less likely to be resolved and identified. The same phenomena happen in the 2DLC setup; additionally, the fast gradient used in the second-dimension chromatography reduces the number of truncated forms that are observable.
RPLC-MS analysis yielded a higher number of H2A and H2B proteoforms, whereas WCX identified a significantly larger number of H3 proteoforms. Notably, the 2DLC setup combines the specificity of both the separations, allowing for the identification of more proteins for all family groups. These results can be explained by the configuration of our 2DLC setup where the 1D (WCX-HILIC) separates protein charge variants using a long and shallow gradient that is fractionated at an interval of 13 min; fractions are sequentially injected on 2D (RPLC) for protein family separations. The mass distribution for the identified proteoforms (Figure S6 of the Supporting Information), as well as the feature maps shown in Supporting Material S3, indicate that methods based on ion exchange (WCX-HILIC and 2DLC) are particularly well suited to resolve H3 forms. For targeted analysis of H2 and/or H4 using the 2DLC platform, it may be necessary to further optimize both separation dimensions. However, 1D RPLC separations, employing long columns and shallow gradients, would likely provide better separation of H2A and H2B proteoforms, which differ slightly in their primary amino acid sequence. (42)
Overall, WCX-HILIC/a×m/RPLC identified more proteoforms in comparison to both 1D approaches. The combination of independent chemically selective separations reduces ion suppression and provides an increased dynamic range of measurements proving the usefulness of such a separation strategy. We speculate that increasing the column length in the first-dimension column and allowing for a longer analysis time and higher sample injection amount would further increase the separation capacity and possibly the data quality obtained.
To further evaluate our results, we performed only absolute mass ProSight searches against the entire human protein database. Analyses with a narrow search window for precursor and fragment ions (10 ppm) were completed in less than 30 min per data set. We used the C score parameter to evaluate the results. This metric measures the level of characterization of a proteoform using a Bayesian approach. (31) For instance, a C score of 3 indicates that two proteoforms in the database can equally well explain the observed data (different combination of PTM), and proteoforms with C scores of 40 or higher are considered to be unambiguously identified. Table 2 shows the distribution of the identification with a C score below 3 (IDs that can be connected with multiple proteoforms), between 3 and 45 (IDs with up to 2 proteoforms), and more than 45 (unique proteoforms).
An absolute mass ProSight search halved the number of unique histone proteoforms identified relative to searches using a restricted database (Table 1), suggesting that a large number of identified proteoforms had not been explicitly included in the annotated database.
The proteoforms identified included several acetylated, (tri-, di-, mono-) methylated, and phosphorylated histone groups. The overlap between identifications (C score >3) obtained using different separation approaches is depicted in Figure 5.

Figure 5

Figure 5. Venn diagram of the histone proteoforms identified with C > 3 using 1DLC RPLC-MS, 1DLC WCX-HILIC-MS, and 2DLC WCX-HILIC/a×m/RPLC-MS (3); Protein IDs available in Table S4 of the Supporting Information.

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The results of this analysis show a large overlap between the identifications from the three techniques employed, with more than 70% of all the identifications being generated from the WCX-HILIC/a×m/RPLC analyses. Finally, the overlap between the proteoforms identified in triplicate analyses (and processed using the same data analysis workflow) accounted for about 40% for WCX-HILIC/a×m/RPLC. The main source of variability between identified proteoforms is the large diversity of H3 proteoforms. This histone family displays a large diversity in the type and location of PTMs, and our results suggest that each analysis may capture only part of this great complexity. Despite improvements in the separation of intact histones enabled by 2DLC, several proteoforms share the same mass coelute. Thus, the quantitative analysis of the data presented can only be attempted monitoring characteristic fragment intensities and requires the development of specific software (48) and was not attempted in our study.

Conclusions

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The vast heterogeneity of histone proteins makes them challenging analytes for top-down MS investigations. This complexity stems from several sources: positional isomers (i.e., different PTM site on the same proteoform), small mass differences between PTMs (e.g., acetylation and trimethylation), and the presence of unknown/unexpected PTMs (e.g., bromination (42)).
Our results demonstrate the high potential of an online nanoflow 2DLC separation for top-down proteomic analyses of histone proteoforms. The separation strength of the proposed method lays in the combination of the chemical selectivity of two orthogonal separation modalities. Herein, the two methods are coupled online to reduce sample losses typical for offline fractionation schemes. In addition, nanoflows were used to reduce sample dilution and enhance MS sensitivity. Ultimately, the combined advantages of this platform allow for the extension of the dynamic range of the MS measurement, increasing the number of confidently identified histone proteoforms relative to 1DLC-MS.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.8b00458.

  • Gradient programming of the 2DLC (Supporting Material S1, Table S1), a schematic illustration of the histone identification workflow (Supporting Material S2, Figure S1). Additional data on 1DLC and 2DLC separations are reported in the Supporting Material S3. This include details of the separation families by 1DLC WCX-HILIC (Figure S2), 1DLC RPLC (Figure S3), 2DLC WCX-HILIC/a×m/RPLC (Figure S4), the TIC of a single modulation of the 2DLC separation (Figure S5), and the analysis of the distribution of neutral masses detected for each histone family (Figure S6). The list of families of proteins identified using WCX-HILIC/a×m/RPLC UVPD-HRMS (Prosight PC 4.0) is collected in Supporting Material S4, Table S2. The results from the triplicate analysis of HeLa Histones using the WCX-HILIC/a×m/RPLC UVPD-HRMS platform are in the Supporting Material S5, Figure S7. In Supporting Materials S6 and S7 are reported examples of proteoform IDs obtained with our analysis workflow using respectively the informed proteomics and Prosight PC data analysis toolkit. Figure S8 shows a detail of Figure 2 reporting the monoisotopic mass area of H3 histones and assignment of H3.2 proteoforms using the informed proteomics workflow and Figure S9–S14 examples of H3.2 proteoform IDs. Figure S15 shows a detail of Figure 2 reporting the monoisotopic mass area of H4 histones and assignment of H4 proteoforms using the informed proteomics workflow and Figure S16–S19 examples of H4 proteoform IDs. Examples of sequence coverage of protein identifications using Prosight PC 4.0 are collected in Supporting Material S7 (PDF)

  • Excel file reporting the output of the Prosight PC 4.0 searches summarized in Table S3 (list of proteoform identified by Prosight PC 4.0 using a restricted database) (XLSX)

  • Excel file reporting the output of the Prosight PC 4.0 searches summarized in Table S4 (list of proteoforms identified using Prosight PC 4.0 using the Human Proteome database and filtered by C score) (XLSX)

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Author Information

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  • Corresponding Author
    • Andrea F. G. Gargano - Center for Analytical Sciences Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands;  Vrije Universiteit Amsterdam, Department of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, de Boelelaan 1085, 1081HV Amsterdam, The Netherlands;  Orcidhttp://orcid.org/0000-0003-3361-7341;  Email: [email protected]
  • Authors
    • Jared B. Shaw - Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States;  Orcidhttp://orcid.org/0000-0002-1130-1728
    • Mowei Zhou - Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States;  Orcidhttp://orcid.org/0000-0003-3575-3224
    • Christopher S. Wilkins - Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
    • Thomas L. Fillmore - Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
    • Ronald J. Moore - Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
    • Govert W. Somsen - Center for Analytical Sciences Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands;  Vrije Universiteit Amsterdam, Department of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, de Boelelaan 1085, 1081HV Amsterdam, The Netherlands
    • Ljiljana Paša-Tolić - Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was financially supported by The Netherlands Organization for Scientific Research by the NWO Veni grant IPA (722.015.009). The authors would like to thank Nikola Tolic and Rui Zhao from the Pacific Northwest National Laboratories as well as Andrei Barcaru and Eva M.O.L. Johansson from the University of Amsterdam for their support and valuable discussions. We gratefully acknowledge Dr. Andrew Alpert (PolyLC inc.) for the support in developing the WCX-HILIC method and Hagen Preik-Steinhoff (VICI-Valco) for the kind gift of the nano HPLC valve. This work was performed at EMSL, a national scientific user facility sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy (DOE). EMSL is located at PNNL, a multidisciplinary national laboratory operated by Battelle for the U.S. DOE.

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  11. 11
    Yuan, Z.-F.; Arnaudo, A. M.; Garcia, B. a. Mass spectrometric analysis of histone proteoforms. Annu. Rev. Anal. Chem. 2014, 7, 113– 128,  DOI: 10.1146/annurev-anchem-071213-015959
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    Mass spectrometric analysis of histone proteoforms
    Yuan, Zuo-Fei; Aranaudo, Anna M.; Garcia, Benjamin A.
    Annual Review of Analytical Chemistry (2014), 7 (), 113-128CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)
    A review. Histories play important roles in chromatin, in the forms of various post-translational modifications (PTMs) and sequence variants, which are called histone proteoforms. Investigating modifications and variants is an ongoing challenge. Previous methods are based on antibodies, and because they usually detect only one modification at a time, they are not suitable for studying the various combinations of modifications on histones. Fortunately, mass spectrometry (MS) has emerged as a high-throughput technol. for histone anal. and does not require prior knowledge about any modifications. From the data generated by mass spectrometers, both identification and quantification of modifications, as well as variants, can be obtained easily. On the basis of this information, the functions of histones in various cellular contexts can be revealed. Therefore, MS continues to play an important role in the study of histone proteoforms. In this review, we discuss the anal. strategies of MS, their applications on histones, and some key remaining challenges.
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  12. 12
    Britton, L.-M. P.; Gonzales-Cope, M.; Zee, B. M.; Garcia, B. A. Breaking the histone code with quantitative mass spectrometry. Expert Rev. Proteomics 2011, 8 (5), 631– 643,  DOI: 10.1586/epr.11.47
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    Breaking the histone code with quantitative mass spectrometry
    Britton, Laura-Mae P.; Gonzales-Cope, Michelle; Zee, Barry M.; Garcia, Benjamin A.
    Expert Review of Proteomics (2011), 8 (5), 631-643CODEN: ERPXA3; ISSN:1478-9450. (Expert Reviews Ltd.)
    A review. Histone post-translational modifications (PTMs) comprise one of the most intricate nuclear signaling networks that govern gene expression in a long-term and dynamic fashion. These PTMs are considered to be epigenetic or heritable from one cell generation to the next and help establish genomic expression patterns. While much of the analyses of histones have historically been performed using site-specific antibodies, these methods are replete with tech. obstacles (i.e., cross-reactivity and epitope occlusion). Mass spectrometry-based proteomics has begun to play a significant role in the interrogation of histone PTMs, revealing many new aspects of these modifications that cannot be easily detd. with std. biol. approaches. Here, we review the accomplishments of mass spectrometry in the histone field, and outline the future roadblocks that must be overcome for mass spectrometry-based proteomics to become the method of choice for chromatin biologists.
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    Smith, L. M.; Kelleher, N. L. Proteoform: a single term describing protein complexity. Nat. Methods 2013, 10 (3), 186– 187,  DOI: 10.1038/nmeth.2369
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    13
    Proteoform: a single term describing protein complexity
    Smith, Lloyd M.; Kelleher, Neil L.; Linial, Michal; Goodlett, David; Langridge-Smith, Pat; Goo, Young Ah; Safford, George; Bonilla, Leo; Kruppa, George; Zubarev, Roman; Rontree, Jon; Chamot-Rooke, Julia; Garavelli, John; Heck, Albert; Loo, Joseph; Penque, Deborah; Hornshaw, Martin; Hendrickson, Christopher; Pasa-Tolic, Ljiljana; Borchers, Christoph; Chan, Dominic; Young, Nicholas; Agar, Jeffrey; Masselon, Christophe; Gross, Michael; McLafferty, Fred; Tsybin, Yury; Ge, Ying; Sanders, Ian; Langridge, James; Whitelegge, Julian; Marshall, Alan
    Nature Methods (2013), 10 (3), 186-187CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    In this article the author proposes that the term proteoform be used to designate all of the different mol. forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications. The term should include all post-translational modifications in the PSI-MOD ontol. except those classified as reagent-derivatized or isotope-labeled residues.
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  14. 14
    Maile, T. M.; Izrael-Tomasevic, A.; Cheung, T.; Guler, G. D.; Tindell, C.; Masselot, A.; Liang, J.; Zhao, F.; Trojer, P.; Classon, M. Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method. Mol. Cell. Proteomics 2015, 14 (4), 1148– 1158,  DOI: 10.1074/mcp.O114.046573
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    14
    Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method
    Maile, Tobias M.; Izrael-Tomasevic, Anita; Cheung, Tommy; Guler, Gulfem D.; Tindell, Charles; Masselot, Alexandre; Liang, Jun; Zhao, Feng; Trojer, Patrick; Classon, Marie; Arnott, David
    Molecular & Cellular Proteomics (2015), 14 (4), 1148-1158CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
    Mass spectrometry is a powerful alternative to antibody-based methods for the anal. of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample prepn. and liq. chromatog.-mass spectrometry. We developed a new method employing a "one-pot" hybrid chem. derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aq. conditions is followed by trypsin digestion and labeling of new peptide N termini with Ph isocyanate. High resoln. mass spectrometry was used to collect qual. and quant. data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 Me, acetyl, and phosphoryl marks on histone H3.1 were improved by an av. of threefold overall, and over 50-fold for H3K4 di- and tri-Me marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quant. changes in H3K4 trimethylation induced by small mol. inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.
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    Zheng, Y.; Huang, X.; Kelleher, N. L. Epiproteomics: Quantitative analysis of histone marks and codes by mass spectrometry. Curr. Opin. Chem. Biol. 2016, 33, 142– 150,  DOI: 10.1016/j.cbpa.2016.06.007
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    Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry
    Zheng, Yupeng; Huang, Xiaoxiao; Kelleher, Neil L.
    Current Opinion in Chemical Biology (2016), 33 (), 142-150CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. Histones are a group of proteins with a high no. of post-translational modifications, including methylation, acetylation, phosphorylation, and monoubiquitination, which play crit. roles in every chromatin-templated activity. The quant. anal. of these modifications using mass spectrometry (MS) has seen significant improvements over the last decade. It is now possible to perform large-scale surveys of dozens of histone marks and hundreds of their combinations on global chromatin. Here, we review the development of three MS strategies for analyzing histone modifications that have come to be known as Bottom Up, Middle Down, and Top Down. We also discuss challenges and innovative solns. for characterizing and quantifying complicated isobaric species arising from multiple modifications on the same histone mol.
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    Molden, R. C.; Garcia, B. A. Middle-down and top-down mass spectrometric analysis of co-occurring histone modifications. Curr. Protoc. Protein Sci. 2014, 77 (1), 23.7.1– 23.7.28,  DOI: 10.1002/0471140864.ps2307s77
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    Tvardovskiy, A.; Wrzesinski, K.; Sidoli, S.; Fey, S. J.; Rogowska-Wrzesinska, A.; Jensen, O. N. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns. Mol. Cell. Proteomics 2015, 14 (12), 3142– 3153,  DOI: 10.1074/mcp.M115.048975
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    Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns
    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone; Fey, Stephen J.; Rogowska-Wrzesinska, Adelina; Jensen, Ole N.
    Molecular & Cellular Proteomics (2015), 14 (12), 3142-3153CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
    Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein anal. by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive anal. of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Anal. of co-existing PTMs revealed neg. crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.
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    Azad, G. K.; Tomar, R. S. Proteolytic clipping of histone tails: The emerging role of histone proteases in regulation of various biological processes. Mol. Biol. Rep. 2014, 41 (5), 2717– 2730,  DOI: 10.1007/s11033-014-3181-y
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    Proteolytic clipping of histone tails: the emerging role of histone proteases in regulation of various biological processes
    Azad, Gajendra Kumar; Tomar, Raghuvir S.
    Molecular Biology Reports (2014), 41 (5), 2717-2730CODEN: MLBRBU; ISSN:0301-4851. (Springer)
    Chromatin is a dynamic DNA scaffold structure that responds to a variety of external and internal stimuli to regulate the fundamental biol. processes. Majority of the cases chromatin dynamicity is exhibited through chem. modifications and phys. changes between DNA and histones. These modifications are reversible and complex signaling pathways involving chromatin-modifying enzymes regulate the fluidity of chromatin. Fluidity of chromatin can also be impacted through irreversible change, proteolytic processing of histones which is a poorly understood phenomenon. In recent studies, histone proteolysis has been implicated as a regulatory process involved in the permanent removal of epigenetic marks from histones. Activities responsible for clipping of histone tails and their significance in various biol. processes have been obsd. in several organisms. Here, we have reviewed the properties of some of the known histone proteases, analyzed their significance in biol. processes and have provided future directions.
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    Greer, S. M.; Brodbelt, J. S. Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry. J. Proteome Res. 2018, 17 (3), 1138– 1145,  DOI: 10.1021/acs.jproteome.7b00801
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    19
    Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry
    Greer, Sylvester M.; Brodbelt, Jennifer S.
    Journal of Proteome Research (2018), 17 (3), 1138-1145CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    The characterization of protein posttranslational modifications (PTMs) remains a significant challenge for traditional bottom-up proteomics methods owing to the lability of PTMs and the difficulty of mapping combinatorial patterns of PTMs based on anal. of small peptides. These shortcomings have accelerated interest in top-down MS/MS methods that focus on anal. of intact proteins. Simultaneous mapping of all PTMs requires extensive sequence coverage to confidently localize modifications. 193 nm UV photodissocn. (UVPD) has been shown to generate unparalleled sequence coverage for intact proteins compared to traditional MS/MS methods. This study focuses on identification and localization of PTMs of histones by UVPD, higher-energy collisional dissocn. (HCD), and the hybrid method electron-transfer/higher-energy collision dissocn. (EThcD) via a high throughput liq. chromatog.-mass spectrometry strategy. In total, over 500 proteoforms were characterized among these three activation methods with 46% of the identifications found in common by two or more activation methods. EThcD and UVPD afforded more extensive characterization of proteoforms than HCD with av. gains in sequence coverage of 15% and C-scores that doubled on av.
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    Wang, T.; Holt, M. V.; Young, N. L. The histone H4 proteoform dynamics in response to SUV4–20 inhibition reveals single molecule mechanisms of inhibitor resistance. Epigenet. Chromatin 2018, 11 (1), 1– 18,  DOI: 10.1186/s13072-018-0198-9
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    Dang, X.; Singh, A.; Spetman, B. D.; Nolan, K. D.; Isaacs, J. S.; Dennis, J. H.; Dalton, S.; Marshall, A. G.; Young, N. L. Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS. J. Proteome Res. 2016, 15 (9), 3196– 3203,  DOI: 10.1021/acs.jproteome.6b00414
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    Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS
    Dang, Xibei; Singh, Amar; Spetman, Brian D.; Nolan, Krystal D.; Isaacs, Jennifer S.; Dennis, Jonathan H.; Dalton, Stephen; Marshall, Alan G.; Young, Nicolas L.
    Journal of Proteome Research (2016), 15 (9), 3196-3203CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resoln. FT-ICR MS and MS/MS yield quant. relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the anal. to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biol. role of many previously unstudied histone variants and post-translational modifications.
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    Toby, T. K.; Fornelli, L.; Kelleher, N. L. Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu. Rev. Anal. Chem. 2016, 9 (1), 499– 519,  DOI: 10.1146/annurev-anchem-071015-041550
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    Progress in Top-Down Proteomics and the Analysis of Proteoforms
    Toby, Timothy K.; Fornelli, Luca; Kelleher, Neil L.
    Annual Review of Analytical Chemistry (2016), 9 (), 499-519CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)
    From a mol. perspective, enactors of function in biol. are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the anal. of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramol. complexity preserved during anal. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissocn. methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss tech. and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clin. and translational research.
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    Tian, Z.; Zhao, R.; Tolić, N.; Moore, R. J.; Stenoien, D. L.; Robinson, E. W.; Smith, R. D.; Paša-Tolić, L. Two-dimensional liquid chromatography system for online top-down mass spectrometry. Proteomics 2010, 10 (20), 3610– 3620,  DOI: 10.1002/pmic.201000367
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    Zhou, M.; Wu, S.; Stenoien, D. L.; Zhang, Z.; Connolly, L.; Freitag, M.; Pasa-Tolic, L. Profiling changes in histone post-translational modifications by top-down mass spectrometry. In Methods Mol. Biol. ; 2017; Vol. 1507, pp 153– 168.
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    Shaw, J. B.; Li, W.; Holden, D. D.; Zhang, Y.; Griep-Raming, J.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation. J. Am. Chem. Soc. 2013, 135 (34), 12646– 12651,  DOI: 10.1021/ja4029654
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    Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation
    Shaw, Jared B.; Li, Wenzong; Holden, Dustin D.; Zhang, Yan; Griep-Raming, Jens; Fellers, Ryan T.; Early, Bryan P.; Thomas, Paul M.; Kelleher, Neil L.; Brodbelt, Jennifer S.
    Journal of the American Chemical Society (2013), 135 (34), 12646-12651CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here the authors describe the implementation of 193 nm UV photodissocn. (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer's disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissocn. methods.
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    Cannon, J. R.; Holden, D. D.; Brodbelt, J. S. Hybridizing ultraviolet photodissociation with electron transfer dissociation for intact protein characterization. Anal. Chem. 2014, 86 (21), 10970– 10977,  DOI: 10.1021/ac5036082
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    Hybridizing Ultraviolet Photodissociation with Electron Transfer Dissociation for Intact Protein Characterization
    Cannon, Joe R.; Holden, Dustin D.; Brodbelt, Jennifer S.
    Analytical Chemistry (Washington, DC, United States) (2014), 86 (21), 10970-10977CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    We report a hybrid fragmentation method involving electron transfer dissocn. (ETD) combined with UV photodissocn. (UVPD) at 193 nm for anal. of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the no. of identified c- and z-type ions obsd. when compared to UVPD or ETD alone, as well as generating a more balanced distribution of a/x, b/y, and c/z ion types. Addnl., the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissocn. (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total no. of fragment ions in comparison to the analogous MS3 format in which ETD and UVPD were undertaken in sep. segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liq. chromatog. timescale characterization was demonstrated for intact ribosomal proteins.
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    Cleland, T. P.; DeHart, C. J.; Fellers, R. T.; Vannispen, A. J.; Greer, J. B.; LeDuc, R. D.; Parker, W. R.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer. J. Proteome Res. 2017, 16 (5), 2072– 2079,  DOI: 10.1021/acs.jproteome.7b00043
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    27
    High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer
    Cleland, Timothy P.; DeHart, Caroline J.; Fellers, Ryan T.; VanNispen, Alexandra J.; Greer, Joseph B.; LeDuc, Richard D.; Parker, W. Ryan; Thomas, Paul M.; Kelleher, Neil L.; Brodbelt, Jennifer S.
    Journal of Proteome Research (2017), 16 (5), 2072-2079CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    The anal. of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by anal. of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. UV photodissocn. (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatog. time scale. However, use of UVPD for high-throughput top-down anal. to date has been limited by bioinformatics. Here the authors detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissocn. (HCD) in a comparative anal. of HeLa whole-cell lysate by qual. top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores >40 for UVPD and 63% for HCD. The av. sequence coverage (28 ± 20% for UVPD vs. 17 ± 8% for HCD, p < 0.0001) is higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.
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    Tian, Z.; Tolić, N.; Zhao, R.; Moore, R. J.; Hengel, S. M.; Robinson, E. W.; Stenoien, D. L.; Wu, S.; Smith, R. D.; Paša-Tolić, L. Enhanced top-down characterization of histone post-translational modifications. Genome Biol. 2012, 13 (10), R86,  DOI: 10.1186/gb-2012-13-10-r86
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    Enhanced top-down characterization of histone post-translational modifications
    Tian, Zhixin; Tolic, Nikola; Zhao, Rui; Moore, Ronald J.; Hengel, Shawna M.; Robinson, Errol W.; Stenoien, David L.; Wu, Si; Smith, Richard D.; Pasa-Tolic, Ljiljana
    Genome Biology (2012), 13 (), R86CODEN: GNBLFW; ISSN:1474-760X. (BioMed Central Ltd.)
    Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liq. chromatog.-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS anal. of 7.5 μg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.
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  29. 29
    Fort, K. L.; Dyachenko, A.; Potel, C. M.; Corradini, E.; Marino, F.; Barendregt, A.; Makarov, A. A.; Scheltema, R. A.; Heck, A. J. R. Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics. Anal. Chem. 2016, 88 (4), 2303– 2310,  DOI: 10.1021/acs.analchem.5b04162
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    29
    Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics
    Fort, Kyle L.; Dyachenko, Andrey; Potel, Clement M.; Corradini, Eleonora; Marino, Fabio; Barendregt, Arjan; Makarov, Alexander A.; Scheltema, Richard A.; Heck, Albert J. R.
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (4), 2303-2310CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissocn. (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biol. questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with UV photodissocn. (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liq. chromatog. (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amts. of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the anal. of post-translational modifications and is a powerful and complementary addn. to the proteomic toolbox.
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  30. 30
    Park, J.; Piehowski, P. D.; Wilkins, C.; Zhou, M.; Mendoza, J.; Fujimoto, G. M.; Gibbons, B. C.; Shaw, J. B.; Shen, Y.; Shukla, A. K. Informed-Proteomics: Open-source software package for top-down proteomics. Nat. Methods 2017, 14 (9), 909– 914,  DOI: 10.1038/nmeth.4388
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    30
    Informed-Proteomics: open-source software package for top-down proteomics
    Park, Jungkap; Piehowski, Paul D.; Wilkins, Christopher; Zhou, Mowei; Mendoza, Joshua; Fujimoto, Grant M.; Gibbons, Bryson C.; Shaw, Jared B.; Shen, Yufeng; Shukla, Anil K.; Moore, Ronald J.; Liu, Tao; Petyuk, Vladislav A.; Tolic, Nikola; Pasa-Tolic, Ljiljana; Smith, Richard D.; Payne, Samuel H.; Kim, Sangtae
    Nature Methods (2017), 14 (9), 909-914CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Top-down proteomics, the anal. of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liq. chromatog.-tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed. Here we present Informed-Proteomics, an open-source software suite for top-down proteomics anal. that consists of an LC-MS feature-finding algorithm, a database search algorithm, and an interactive results viewer. We compare our tool with several other popular tools using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up anal.
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  31. 31
    Leduc, R. D.; Fellers, R. T.; Early, B. P.; Greer, J. B.; Thomas, P. M.; Kelleher, N. L. The C-Score: A bayesian framework to sharply improve proteoform scoring in high-throughput top down proteomics. J. Proteome Res. 2014, 13 (7), 3231– 3240,  DOI: 10.1021/pr401277r
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    31
    The C-Score: A Bayesian Framework to Sharply Improve Proteoform Scoring in High-Throughput Top Down Proteomics
    LeDuc, Richard D.; Fellers, Ryan T.; Early, Bryan P.; Greer, Joseph B.; Thomas, Paul M.; Kelleher, Neil L.
    Journal of Proteome Research (2014), 13 (7), 3231-3240CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    The automated processing of data generated by top down proteomics would benefit from improved scoring for protein identification and characterization of highly related protein forms (proteoforms). Here the authors propose the "C-score" (short for Characterization Score), a Bayesian approach to the proteoform identification and characterization problem, implemented within a framework to allow the infusion of expert knowledge into generative models that take advantage of known properties of proteins and top down anal. systems (e.g., fragmentation propensities, "off-by-1 Da" discontinuous errors, and intelligent weighting for site-specific modifications). The performance of the scoring system based on the initial generative models was compared to the current probability-based scoring system used within both ProSightPC and ProSightPTM on a manually curated set of 295 human proteoforms. The current implementation of the C-score framework generated a marked improvement over the existing scoring system as measured by the area under the curve on the resulting ROC chart (AUC of 0.99 vs. 0.78).
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    Lindner, H.; Sarg, B.; Meraner, C.; Helliger, W. Separation of acetylated core histones by hydrophilic-interaction liquid chromatography. J. Chromatogr. A 1996, 743 (1), 137– 144,  DOI: 10.1016/0021-9673(96)00131-8
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    32
    Separation of acetylated core histones by hydrophilic-interaction liquid chromatography
    Lindner, Herbert; Sarg, Bettina; Meraner, Christoph; Helliger, Wilfried
    Journal of Chromatography A (1996), 743 (1), 137-144CODEN: JCRAEY; ISSN:0021-9673. (Elsevier)
    Hydrophilic-interaction liq. chromatog. (HILIC) has recently been introduced as a highly efficient chromatog. technique for the sepn. of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the sepn. of posttranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, vol./vol.) at pH 3.0. In combination with reversed-phase-high performance liq. chromatog. (RP-HPLC) we have successfully sepd. various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the anal. and isolation of modified core histone variants HILIC provides a new and important alternative to traditional sepn. techniques and will be useful in studying the biol. function of histone acetylation.
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    Lindner, H.; Sarg, B.; Helliger, W. Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones. J. Chromatogr. A 1997, 782 (1), 55– 62,  DOI: 10.1016/S0021-9673(97)00468-8
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    Benevento, M.; Tonge, P. D.; Puri, M. C.; Nagy, A.; Heck, A. J. R.; Munoz, J. Fluctuations in histone H4 isoforms during cellular reprogramming monitored by middle-down proteomics. Proteomics 2015, 15 (18), 3219– 3231,  DOI: 10.1002/pmic.201500031
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    Fluctuations in histone H4 isoforms during cellular reprogramming monitored by middle-down proteomics
    Benevento, Marco; Tonge, Peter D.; Puri, Mira C.; Nagy, Andras; Heck, Albert J. R.; Munoz, Javier
    Proteomics (2015), 15 (18), 3219-3231CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somatic cells into an embryonic-like pluripotent state. Post-translational modifications of histones play an important role in this process. Previously, we found by ChIP-seq widespread changes of specific histone H3 marks in two divergent reprogramming routes leading to alternative pluripotent sates . Here, using an unbiased middle-down proteomics approach we have identified 72 unique isoforms of histone H4 and quantified 56 of them in the same set of samples. We found substantial differences between somatic and late-phase reprogramming cells. Also, ESCs and iPSCs displayed higher levels of H4 acetylation and tri-methylation concomitantly with lower levels of mono- and di-methylation when compared to cells undergoing reprogramming. Our data shows that the epigenetic remodeling induced by the reprogramming process goes beyond histone H3 and reveals the importance of H4 modifications as well. The presented data is a valuable resource to study the epigenetic mechanisms involved in the acquisition of induced pluripotency. All MS data have been deposited in the ProteomeXchange with identifier PXD002062.
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    Young, N. L.; DiMaggio, P. a.; Plazas-Mayorca, M. D.; Baliban, R. C.; Floudas, C. A.; Garcia, B. A. High throughput characterization of combinatorial histone codes. Mol. Cell. Proteomics 2009, 8 (10), 2266– 2284,  DOI: 10.1074/mcp.M900238-MCP200
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    High throughput characterization of combinatorial histone codes
    Young, Nicolas L.; Di Maggio, Peter A.; Plazas-Mayorca, Mariana D.; Baliban, Richard C.; Floudas, Christodoulos A.; Garcia, Benjamin A.
    Molecular and Cellular Proteomics (2009), 8 (10), 2266-2284CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)
    We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liq. chromatog. directly coupled to electron transfer dissocn. (ETD) mass spectrometry for the automated online high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resoln. mass spectrometer, displays an improvement over existing methods with an ∼100-fold redn. in sample requirements and anal. time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h anal. The large N-terminal histone peptides are eluted by the pH and org. solvent weak cation exchange-hydrophilic interaction liq. chromatog. gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric tri-Me and acetyl modifications are resolved chromatog. and confidently distinguished by the synthesis of mass spectrometric and chromatog. information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the anal. of highly modified peptides. We find this methodol. very useful for chromatog. sepn. of isomeric species that cannot be sepd. well by any other chromatog. means, leading to less complicated tandem mass spectra. The improved sepn. and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3.2 forms and 70 H4 forms, including forms not yet detected in human cells, such as the remarkably highly modified histone H3.2 K4me3K9acK14acK18acK23acK27acK36me3. Such detail provided by our proteomics platform will be essential for detg. how histone modifications occur and act in combination to propagate the histone code during transcriptional events and could greatly enable sequencing of the histone component of human epigenomes.
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    Jung, H. R.; Sidoli, S.; Haldbo, S.; Sprenger, R. R.; Schwämmle, V.; Pasini, D.; Helin, K.; Jensen, O. N. Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS. Anal. Chem. 2013, 85 (17), 8232– 8239,  DOI: 10.1021/ac401299w
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    Precision Mapping of Coexisting Modifications in Histone H3 Tails from Embryonic Stem Cells by ETD-MS/MS
    Jung, Hye Ryung; Sidoli, Simone; Haldbo, Simon; Sprenger, Richard R.; Schwammle, Veit; Pasini, Diego; Helin, Kristian; Jensen, Ole N.
    Analytical Chemistry (Washington, DC, United States) (2013), 85 (17), 8232-8239CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, the authors report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone H3 N-terminal tail peptides (amino acids 1-50, 5-6 kDa) were sepd. by optimized weak cation exchange/hydrophilic interaction liq. chromatog. (WCX/HILIC) and sequenced online by electron transfer dissocn. (ETD) tandem mass spectrometry (MS/MS). High mass accuracy and near complete sequence coverage allowed unambiguous mapping of the major histone marks and discrimination between isobaric and nearly isobaric PTMs such as trimethylation and acetylation. Hierarchical data anal. identified H3K27me2-H3K36me2 as the most frequently obsd. PTMs in H3. Modifications at H3 residues K27 and K36 often coexist with the abundant mark K23ac, and the authors identified two frequently occurring quadruplet marks K9me1K23acK27me2K36me2 and K9me3K23acK27me2K36me, which might indicate a role in crosstalk. Co-occurrence frequency anal. revealed also an interplay between methylations of K9, K27, and K36, suggesting interdependence between histone methylation marks. The authors hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells.
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    Mizzen, C. A.; Alpert, A. J.; Lévesque, L.; Kruck, T. P. A.; McLachlan, D. R. Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography. J. Chromatogr., Biomed. Appl. 2000, 744 (1), 33– 46,  DOI: 10.1016/S0378-4347(00)00210-3
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    Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography
    Mizzen, C. A.; Alpert, A. J.; Levesque, L.; Kruck, T. P. A.; McLachlan, D. R.
    Journal of Chromatography B: Biomedical Sciences and Applications (2000), 744 (1), 33-46CODEN: JCBBEP; ISSN:0378-4347. (Elsevier Science B.V.)
    A mixed-mode high-performance liq. chromatog. (HPLC) method that resolves the six known non-allelic variants of chicken erythrocyte histone H1 is described. Common, but previously unknown, allelic variants of H1 that co-migrate in PAGE are also resolved. The resoln. of H1 variants achieved by this method should be useful in detg. the functional significance of H1 sequence heterogeneity and in analyses of post-translational modification of H1. Furthermore, the principles behind the sepn. should be applicable to analyses of polymorphism in other proteins.
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    Papazyan, R.; Taverna, S. D. Separation and Purification of Multiply Acetylated Proteins Using Cation-Exchange Chromatography. Methods Mol. Biol. 2013, 981, 103– 113,  DOI: 10.1007/978-1-62703-305-3_8
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    Separation and purification of multiply acetylated proteins using cation-exchange chromatography
    Papazyan, Romeo; Taverna, Sean D.
    Methods in Molecular Biology (New York, NY, United States) (2013), 981 (Protein Acetylation), 103-113CODEN: MMBIED; ISSN:1064-3745. (Springer)
    High-performance liq. chromatog. (HPLC) is extremely useful for the study of proteins and the characterization of their posttranslational modifications. Here we describe a method that utilizes cation-exchange HPLC to sep. multiply acetylated histone H3 species on the basis of their charge and hydrophilicity. This high-resoln. method allows for the sepn. of histone H3 species that differ by as few as one acetyl group, and is compatible with subsequent anal. by a variety of techniques, including mass spectrometry and western blotting.
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    Lindner, H. H. Analysis of histones, histone variants, and their post-translationally modified forms. Electrophoresis 2008, 29 (12), 2516– 2532,  DOI: 10.1002/elps.200800094
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    Analysis of histones, histone variants, and their post-translationally modified forms
    Lindner, Herbert H.
    Electrophoresis (2008), 29 (12), 2516-2532CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. For many years, histones were considered passive structural components of eukaryotic chromatin. Meanwhile it has been proven that histones also participate in gene regulation and repression via post-translational modification. The multitude of these post-translational modifications and the existence of numerous histone variants require particular sepn. strategies for their anal., a prerequisite for studying biol. processes. The most widely utilized techniques for the sepn. of histones, namely PAGE, HPCE, RP-HPLC, and hydrophilic Interaction LC, are reviewed here. Problems inherent to the anal. of histones owing to their unique phys. and chem. properties along with advantages and shortcomings of particular methods are discussed.
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    Zheng, Y.; Fornelli, L.; Compton, P. D.; Sharma, S.; Canterbury, J.; Mullen, C.; Zabrouskov, V.; Fellers, R. T.; Thomas, P. M.; Licht, J. D. Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression. Mol. Cell. Proteomics 2016, 16 (18), 1– 40,  DOI: 10.1074/mcp.M115.053819
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    Garcia, B. A.; Thomas, C. E.; Kelleher, N. L.; Mizzen, C. A. Tissue-specific expression and post-translational modification of histone H3 variants. J. Proteome Res. 2008, 7 (10), 4225– 4236,  DOI: 10.1021/pr800044q
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    Tissue-Specific Expression and Post-Translational Modification of Histone H3 Variants
    Garcia, Benjamin A.; Thomas, C. Eric; Kelleher, Neil L.; Mizzen, Craig A.
    Journal of Proteome Research (2008), 7 (10), 4225-4236CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Analyses of histone H3 from 10 rat tissues using a Middle Down proteomics platform revealed tissue-specific differences in their expression and global PTM abundance. ESI/FTMS with electron capture dissocn. showed that, in general, these proteins were hypomodified in heart, liver and testes. H3.3 was hypermodified compared to H3.2 in some, but not all tissues. In addn., a novel rat testes-specific H3 protein was identified with this approach.
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    Zhou, M.; Paša-Tolić, L.; Stenoien, D. L. Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry. J. Proteome Res. 2017, 16 (2), 599– 608,  DOI: 10.1021/acs.jproteome.6b00694
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    Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry
    Zhou, Mowei; Pasa-Tolic, Ljiljana; Stenoien, David L.
    Journal of Proteome Research (2017), 16 (2), 599-608CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biol. and their roles in disease development have been the subject of intense study. Since multiple PTMs along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone anal. primarily because of deficiencies in sepn./resolving power and effective identification algorithms. Here, the authors used state of the art mass spectrometry and a bioinformatics workflow for targeted data anal. and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.
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    Su, X.; Jacob, N. K.; Amunugama, R.; Lucas, D. M.; Knapp, A. R.; Ren, C.; Davis, M. E.; Marcucci, G.; Parthun, M. R.; Byrd, J. C. Liquid chromatography mass spectrometry profiling of histones. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 850 (1–2), 440– 454,  DOI: 10.1016/j.jchromb.2006.12.037
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    Liquid chromatography mass spectrometry profiling of histones
    Su, Xiaodan; Jacob, Naduparambil K.; Amunugama, Ravindra; Lucas, David M.; Knapp, Amy R.; Ren, Chen; Davis, Melanie E.; Marcucci, Guido; Parthun, Mark R.; Byrd, John C.; Fishel, Richard; Freitas, Michael A.
    Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2007), 850 (1-2), 440-454CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
    Here the authors describe the use of reverse-phase liq. chromatog. mass spectrometry (RPLC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The anal. of intact proteins significantly reduces the time of sample prepn. and simplifies data interpretation. LC-MS anal. and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS anal. allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the anal. of histone H2A species using RPLC fractionation, AU-PAGE sepn. and nano-LC-MS/MS.
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    Pesavento, J. J.; Bullock, C. R.; LeDuc, R. D.; Mizzen, C. A.; Kelleher, N. L. Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry. J. Biol. Chem. 2008, 283 (22), 14927– 14937,  DOI: 10.1074/jbc.M709796200
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    Combinatorial Modification of Human Histone H4 Quantitated by Two-dimensional Liquid Chromatography Coupled with Top Down Mass Spectrometry
    Pesavento, James J.; Bullock, Courtney R.; LeDuc, Richard D.; Mizzen, Craig A.; Kelleher, Neil L.
    Journal of Biological Chemistry (2008), 283 (22), 14927-14937CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Quant. proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and abs. quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >104 using a strictly Top Down mass spectrometric approach coupled with two dimensions of liq. chromatog. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quant. analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quant. anal. This work represents the most comprehensive anal. of histone H4 forms present in human cells reported to date.
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    Vonk, R. J.; Gargano, A. F. G.; Davydova, E.; Dekker, H. L.; Eeltink, S.; de Koning, L. J.; Schoenmakers, P. J. Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae. Anal. Chem. 2015, 87 (10), 5387– 5394,  DOI: 10.1021/acs.analchem.5b00708
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    Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae
    Vonk, Rudy J.; Gargano, Andrea F. G.; Davydova, Ekaterina; Dekker, Henk L.; Eeltink, Sebastiaan; de Koning, Leo J.; Schoenmakers, Peter J.
    Analytical Chemistry (Washington, DC, United States) (2015), 87 (10), 5387-5394CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Stationary-phase-assisted modulation was used to overcome one of the limitations of contemporary comprehensive two-dimensional liq. chromatog., which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high diln. of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. Stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diams. without loss in system performance. In the current setup, a 0.30 mm internal diam. first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diam. second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resoln. mass spectrometer and applied for the sepn. of the tryptic peptides of a six-protein mixt. and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, ∼20000 MS/MS spectra were generated within 24 h anal. time, resulting in the identification of 701 proteins.
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    Yuan, Z. F.; Sidoli, S.; Marchione, D. M.; Simithy, J.; Janssen, K. A.; Szurgot, M. R.; Garcia, B. A. EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data. J. Proteome Res. 2018, 17 (7), 2533– 2541,  DOI: 10.1021/acs.jproteome.8b00133
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    EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data
    Yuan, Zuo-Fei; Sidoli, Simone; Marchione, Dylan M.; Simithy, Johayra; Janssen, Kevin A.; Szurgot, Mary R.; Garcia, Benjamin A.
    Journal of Proteome Research (2018), 17 (7), 2533-2541CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Epigenetics has become a fundamental scientific discipline with various implications for biol. and medicine. Epigenetic marks, mostly DNA methylation and histone posttranslational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here the authors present EpiProfile 2.0, an extended version of the 2015 software (v1.0), for accurate quantification of histone peptides based on liq. chromatog.-tandem mass spectrometry (LC-MS/MS) anal. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extd. ion chromatog. to accurately det. the chromatog. profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, the authors anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biol. and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family.
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  • Abstract

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    Figure 1

    Figure 1. TIC LC-MS chromatogram (top) and feature map (deconvoluted mass spectra vs time) LC-MS chromatograms of 1DLC analysis (a) WCX-HILIC run and (b) RPLC MS. The colored areas indicate the elution zone of the different histone groups.

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    Figure 2

    Figure 2. (a) Schematic representation of the 2DLC-MS/MS setup (WCX-HILIC/a×m/RPLC UVPD-HRMS). The sample components are separated using a gradient from 70% ACN 1%FA (A1) to 68% ACN 8% FA (B1; more details about the gradient programming are reported in the Experimental Section) on a WCX column, fractionated (online) using two trap columns (C18) and sequentially separated using a RPLC column (C18) with a water (A1) to ACN gradient (A2). During the analysis a dilution flow is delivered to reduce the elution strength of the mobile phase of the 1D (dilution flow rate ≈ 9× 1D flow rate) and allow the concentration of the analytes on a trap column (T1). Once the valve is switched, the analytes are eluted from the trap and separated on the 2D column. (b) Diagram reporting the gradient programmed for the 1D (WCX-HILIC) and 2D (RPLC); the green lines mark the injection events on the 1D column, while the blu and red trace the 1D and 2D gradient, respectively.

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    Figure 3

    Figure 3. WCX-HILIC/a×m/RPLC UVPD-HRMS analysis of HeLa core histones. (a) Folded 2D chromatogram extracted from the total ion chromatograms (TICs). 2DLC separation combines the charge resolving power from the WCX separation with the histone family group separation (H2, H3, H4) afforded by RPLC. (b) TICs (top) of the 2DLC separation and the respective neutral mass spectra (bottom).

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    Figure 4

    Figure 4. Detailed view extracted from Figure 3 showing the neutral mass region of H3 (a) and H4 (b) histones. Species with a higher degree of acetylation (Ac) (i.e., with higher molecular mass) have lower retention in WCX-HILIC. Information on histone identifications is provided in Supporting Material S6.

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    Figure 5

    Figure 5. Venn diagram of the histone proteoforms identified with C > 3 using 1DLC RPLC-MS, 1DLC WCX-HILIC-MS, and 2DLC WCX-HILIC/a×m/RPLC-MS (3); Protein IDs available in Table S4 of the Supporting Information.

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  • References

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      Molecular and Cellular Proteomics (2012), 11 (5), 128-137CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
      The defining step in most chromatin immunopptn. (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state assocd. with segments of chromatin. The specificity of the antibody is crit. to the interpretation of the expt., yet this property is rarely reported. Here, we present a quant. method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code.". We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixt. of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, com. available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster anal., we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP expts. need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biol. interpretation exist on the same histone protein mol., and 3) some components of the histone code may be transduced on single proteins in living cells.
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    7. 7
      Contrepois, K.; Ezan, E.; Mann, C.; Fenaille, F. Ultra-high performance liquid chromatography-mass spectrometry for the fast profiling of histone post-translational modifications. J. Proteome Res. 2010, 9 (10), 5501– 5509,  DOI: 10.1021/pr100497a
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      Ultra-High Performance Liquid Chromatography-Mass Spectrometry for the Fast Profiling of Histone Post-Translational Modifications
      Contrepois, Kevin; Ezan, Eric; Mann, Carl; Fenaille, Francois
      Journal of Proteome Research (2010), 9 (10), 5501-5509CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Histones are subjected to extensive post-translational modifications (PTMs) that are known to play key roles in many biol. processes. In this study, the authors report a fast, efficient, highly reproducible, and easily automated method involving ultra-high performance liq. chromatog. (UHPLC) coupled to a high resoln./high mass accuracy LTQ-Orbitrap mass spectrometer to profile core histone modifications/variants from WI-38 primary human fibroblasts. The whole anal. was performed on intact unfractionated histones within 19 min, which is ∼3-fold faster than previously published procedures. High mass accuracy measurements combined with top-down tandem mass spectrometry (MS) expts. enable accurate histone identification. Exptl. and biol. variations were thoroughly assessed and were 8% and 16% on av., resp. With a sample prepn. reduced to the min., characterization of the most abundant histones can be achieved in a single expt. Semi-quant. information can be obtained with respect to the relative abundances of the detected isoforms through a label-free approach. Isoform identities and relative distributions were further confirmed by the LC-MS/MS anal. of tryptic digests. Overall, the authors' UHPLC-MS approach for histone profiling offers a sensitive and reproducible tool that will be of great value for exploring PTMs and variants and can readily be applied to clin. or pharmaceutical studies.
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    8. 8
      Liu, X.; Hengel, S.; Wu, S.; Tolic, N.; Pasa-Tolic, L.; Pevzner, P. A. Identification of ultramodified proteins using top-down tandem mass spectra. J. Proteome Res. 2013, 12 (12), 5830– 5838,  DOI: 10.1021/pr400849y
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      Identification of Ultramodified Proteins Using Top-Down Tandem Mass Spectra
      Liu, Xiaowen; Hengel, Shawna; Wu, Si; Tolic, Nikola; Pasa-Tolic, Ljiljana; Pevzner, Pavel A.
      Journal of Proteome Research (2013), 12 (12), 5830-5838CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Post-translational modifications (PTMs) play an important role in various biol. processes through changing protein structure and function. Some ultramodified proteins (like histones) have multiple PTMs forming PTM patterns that define the functionality of a protein. While bottom-up mass spectrometry (MS) has been successful in identifying individual PTMs within short peptides, it is unable to identify PTM patterns spreading along entire proteins in a coordinated fashion. In contrast, top-down MS analyzes intact proteins and reveals PTM patterns along the entire proteins. However, while recent advances in instrumentation have made top-down MS accessible to many labs., most computational tools for top-down MS focus on proteins with few PTMs and are unable to identify complex PTM patterns. We propose a new algorithm, MS-Align-E, that identifies both expected and unexpected PTMs in ultramodified proteins. We demonstrate that MS-Align-E identifies many proteoforms of histone H4 and benchmark it against the currently accepted software tools.
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      Baker, M. Mass spectrometry for chromatin biology. Nat. Methods 2012, 9 (7), 649– 652,  DOI: 10.1038/nmeth.2081
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      Mass spectrometry for chromatin biology
      Baker, Monya
      Nature Methods (2012), 9 (7_part1), 649-652CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      A review. To discover new histone marks and interactions, researchers turn to the sophisticated instruments of proteomics.
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      Garcia, B. A.; Mollah, S.; Ueberheide, B. M.; Busby, S. A.; Muratore, T. L.; Shabanowitz, J.; Hunt, D. F. Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat. Protoc. 2007, 2 (4), 933– 938,  DOI: 10.1038/nprot.2007.106
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      Chemical derivatization of histones for facilitated analysis by mass spectrometry
      Garcia, Benjamin A.; Mollah, Sahana; Ueberheide, Beatrix M.; Busby, Scott A.; Muratore, Tara L.; Shabanowitz, Jeffrey; Hunt, Donald F.
      Nature Protocols (2007), 2 (4), 933-938CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, the authors describe protocols for the characterization of histone modifications in both qual. and semiquant. manners using chem. derivatization and tandem mass spectrometry. In these procedures, extd. histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addn., a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodol. has the advantage of producing a small no. of predicted peptides from highly modified proteins. The protocol should take approx. 15-19 h to complete, including all chem. reactions, enzymic digestion and mass spectrometry expts.
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      Yuan, Z.-F.; Arnaudo, A. M.; Garcia, B. a. Mass spectrometric analysis of histone proteoforms. Annu. Rev. Anal. Chem. 2014, 7, 113– 128,  DOI: 10.1146/annurev-anchem-071213-015959
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      Mass spectrometric analysis of histone proteoforms
      Yuan, Zuo-Fei; Aranaudo, Anna M.; Garcia, Benjamin A.
      Annual Review of Analytical Chemistry (2014), 7 (), 113-128CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)
      A review. Histories play important roles in chromatin, in the forms of various post-translational modifications (PTMs) and sequence variants, which are called histone proteoforms. Investigating modifications and variants is an ongoing challenge. Previous methods are based on antibodies, and because they usually detect only one modification at a time, they are not suitable for studying the various combinations of modifications on histones. Fortunately, mass spectrometry (MS) has emerged as a high-throughput technol. for histone anal. and does not require prior knowledge about any modifications. From the data generated by mass spectrometers, both identification and quantification of modifications, as well as variants, can be obtained easily. On the basis of this information, the functions of histones in various cellular contexts can be revealed. Therefore, MS continues to play an important role in the study of histone proteoforms. In this review, we discuss the anal. strategies of MS, their applications on histones, and some key remaining challenges.
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      Britton, L.-M. P.; Gonzales-Cope, M.; Zee, B. M.; Garcia, B. A. Breaking the histone code with quantitative mass spectrometry. Expert Rev. Proteomics 2011, 8 (5), 631– 643,  DOI: 10.1586/epr.11.47
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      Breaking the histone code with quantitative mass spectrometry
      Britton, Laura-Mae P.; Gonzales-Cope, Michelle; Zee, Barry M.; Garcia, Benjamin A.
      Expert Review of Proteomics (2011), 8 (5), 631-643CODEN: ERPXA3; ISSN:1478-9450. (Expert Reviews Ltd.)
      A review. Histone post-translational modifications (PTMs) comprise one of the most intricate nuclear signaling networks that govern gene expression in a long-term and dynamic fashion. These PTMs are considered to be epigenetic or heritable from one cell generation to the next and help establish genomic expression patterns. While much of the analyses of histones have historically been performed using site-specific antibodies, these methods are replete with tech. obstacles (i.e., cross-reactivity and epitope occlusion). Mass spectrometry-based proteomics has begun to play a significant role in the interrogation of histone PTMs, revealing many new aspects of these modifications that cannot be easily detd. with std. biol. approaches. Here, we review the accomplishments of mass spectrometry in the histone field, and outline the future roadblocks that must be overcome for mass spectrometry-based proteomics to become the method of choice for chromatin biologists.
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      Smith, L. M.; Kelleher, N. L. Proteoform: a single term describing protein complexity. Nat. Methods 2013, 10 (3), 186– 187,  DOI: 10.1038/nmeth.2369
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      Proteoform: a single term describing protein complexity
      Smith, Lloyd M.; Kelleher, Neil L.; Linial, Michal; Goodlett, David; Langridge-Smith, Pat; Goo, Young Ah; Safford, George; Bonilla, Leo; Kruppa, George; Zubarev, Roman; Rontree, Jon; Chamot-Rooke, Julia; Garavelli, John; Heck, Albert; Loo, Joseph; Penque, Deborah; Hornshaw, Martin; Hendrickson, Christopher; Pasa-Tolic, Ljiljana; Borchers, Christoph; Chan, Dominic; Young, Nicholas; Agar, Jeffrey; Masselon, Christophe; Gross, Michael; McLafferty, Fred; Tsybin, Yury; Ge, Ying; Sanders, Ian; Langridge, James; Whitelegge, Julian; Marshall, Alan
      Nature Methods (2013), 10 (3), 186-187CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      In this article the author proposes that the term proteoform be used to designate all of the different mol. forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and post-translational modifications. The term should include all post-translational modifications in the PSI-MOD ontol. except those classified as reagent-derivatized or isotope-labeled residues.
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      Maile, T. M.; Izrael-Tomasevic, A.; Cheung, T.; Guler, G. D.; Tindell, C.; Masselot, A.; Liang, J.; Zhao, F.; Trojer, P.; Classon, M. Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method. Mol. Cell. Proteomics 2015, 14 (4), 1148– 1158,  DOI: 10.1074/mcp.O114.046573
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      Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method
      Maile, Tobias M.; Izrael-Tomasevic, Anita; Cheung, Tommy; Guler, Gulfem D.; Tindell, Charles; Masselot, Alexandre; Liang, Jun; Zhao, Feng; Trojer, Patrick; Classon, Marie; Arnott, David
      Molecular & Cellular Proteomics (2015), 14 (4), 1148-1158CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
      Mass spectrometry is a powerful alternative to antibody-based methods for the anal. of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample prepn. and liq. chromatog.-mass spectrometry. We developed a new method employing a "one-pot" hybrid chem. derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aq. conditions is followed by trypsin digestion and labeling of new peptide N termini with Ph isocyanate. High resoln. mass spectrometry was used to collect qual. and quant. data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 Me, acetyl, and phosphoryl marks on histone H3.1 were improved by an av. of threefold overall, and over 50-fold for H3K4 di- and tri-Me marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quant. changes in H3K4 trimethylation induced by small mol. inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.
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      Zheng, Y.; Huang, X.; Kelleher, N. L. Epiproteomics: Quantitative analysis of histone marks and codes by mass spectrometry. Curr. Opin. Chem. Biol. 2016, 33, 142– 150,  DOI: 10.1016/j.cbpa.2016.06.007
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      Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry
      Zheng, Yupeng; Huang, Xiaoxiao; Kelleher, Neil L.
      Current Opinion in Chemical Biology (2016), 33 (), 142-150CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Histones are a group of proteins with a high no. of post-translational modifications, including methylation, acetylation, phosphorylation, and monoubiquitination, which play crit. roles in every chromatin-templated activity. The quant. anal. of these modifications using mass spectrometry (MS) has seen significant improvements over the last decade. It is now possible to perform large-scale surveys of dozens of histone marks and hundreds of their combinations on global chromatin. Here, we review the development of three MS strategies for analyzing histone modifications that have come to be known as Bottom Up, Middle Down, and Top Down. We also discuss challenges and innovative solns. for characterizing and quantifying complicated isobaric species arising from multiple modifications on the same histone mol.
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      Molden, R. C.; Garcia, B. A. Middle-down and top-down mass spectrometric analysis of co-occurring histone modifications. Curr. Protoc. Protein Sci. 2014, 77 (1), 23.7.1– 23.7.28,  DOI: 10.1002/0471140864.ps2307s77
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      Tvardovskiy, A.; Wrzesinski, K.; Sidoli, S.; Fey, S. J.; Rogowska-Wrzesinska, A.; Jensen, O. N. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns. Mol. Cell. Proteomics 2015, 14 (12), 3142– 3153,  DOI: 10.1074/mcp.M115.048975
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      Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns
      Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone; Fey, Stephen J.; Rogowska-Wrzesinska, Adelina; Jensen, Ole N.
      Molecular & Cellular Proteomics (2015), 14 (12), 3142-3153CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)
      Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein anal. by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive anal. of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Anal. of co-existing PTMs revealed neg. crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.
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      Azad, G. K.; Tomar, R. S. Proteolytic clipping of histone tails: The emerging role of histone proteases in regulation of various biological processes. Mol. Biol. Rep. 2014, 41 (5), 2717– 2730,  DOI: 10.1007/s11033-014-3181-y
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      Proteolytic clipping of histone tails: the emerging role of histone proteases in regulation of various biological processes
      Azad, Gajendra Kumar; Tomar, Raghuvir S.
      Molecular Biology Reports (2014), 41 (5), 2717-2730CODEN: MLBRBU; ISSN:0301-4851. (Springer)
      Chromatin is a dynamic DNA scaffold structure that responds to a variety of external and internal stimuli to regulate the fundamental biol. processes. Majority of the cases chromatin dynamicity is exhibited through chem. modifications and phys. changes between DNA and histones. These modifications are reversible and complex signaling pathways involving chromatin-modifying enzymes regulate the fluidity of chromatin. Fluidity of chromatin can also be impacted through irreversible change, proteolytic processing of histones which is a poorly understood phenomenon. In recent studies, histone proteolysis has been implicated as a regulatory process involved in the permanent removal of epigenetic marks from histones. Activities responsible for clipping of histone tails and their significance in various biol. processes have been obsd. in several organisms. Here, we have reviewed the properties of some of the known histone proteases, analyzed their significance in biol. processes and have provided future directions.
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      Greer, S. M.; Brodbelt, J. S. Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry. J. Proteome Res. 2018, 17 (3), 1138– 1145,  DOI: 10.1021/acs.jproteome.7b00801
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      19
      Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry
      Greer, Sylvester M.; Brodbelt, Jennifer S.
      Journal of Proteome Research (2018), 17 (3), 1138-1145CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The characterization of protein posttranslational modifications (PTMs) remains a significant challenge for traditional bottom-up proteomics methods owing to the lability of PTMs and the difficulty of mapping combinatorial patterns of PTMs based on anal. of small peptides. These shortcomings have accelerated interest in top-down MS/MS methods that focus on anal. of intact proteins. Simultaneous mapping of all PTMs requires extensive sequence coverage to confidently localize modifications. 193 nm UV photodissocn. (UVPD) has been shown to generate unparalleled sequence coverage for intact proteins compared to traditional MS/MS methods. This study focuses on identification and localization of PTMs of histones by UVPD, higher-energy collisional dissocn. (HCD), and the hybrid method electron-transfer/higher-energy collision dissocn. (EThcD) via a high throughput liq. chromatog.-mass spectrometry strategy. In total, over 500 proteoforms were characterized among these three activation methods with 46% of the identifications found in common by two or more activation methods. EThcD and UVPD afforded more extensive characterization of proteoforms than HCD with av. gains in sequence coverage of 15% and C-scores that doubled on av.
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      Wang, T.; Holt, M. V.; Young, N. L. The histone H4 proteoform dynamics in response to SUV4–20 inhibition reveals single molecule mechanisms of inhibitor resistance. Epigenet. Chromatin 2018, 11 (1), 1– 18,  DOI: 10.1186/s13072-018-0198-9
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      Dang, X.; Singh, A.; Spetman, B. D.; Nolan, K. D.; Isaacs, J. S.; Dennis, J. H.; Dalton, S.; Marshall, A. G.; Young, N. L. Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS. J. Proteome Res. 2016, 15 (9), 3196– 3203,  DOI: 10.1021/acs.jproteome.6b00414
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      21
      Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS
      Dang, Xibei; Singh, Amar; Spetman, Brian D.; Nolan, Krystal D.; Isaacs, Jennifer S.; Dennis, Jonathan H.; Dalton, Stephen; Marshall, Alan G.; Young, Nicolas L.
      Journal of Proteome Research (2016), 15 (9), 3196-3203CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resoln. FT-ICR MS and MS/MS yield quant. relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the anal. to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biol. role of many previously unstudied histone variants and post-translational modifications.
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      Toby, T. K.; Fornelli, L.; Kelleher, N. L. Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu. Rev. Anal. Chem. 2016, 9 (1), 499– 519,  DOI: 10.1146/annurev-anchem-071015-041550
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      Progress in Top-Down Proteomics and the Analysis of Proteoforms
      Toby, Timothy K.; Fornelli, Luca; Kelleher, Neil L.
      Annual Review of Analytical Chemistry (2016), 9 (), 499-519CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)
      From a mol. perspective, enactors of function in biol. are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the anal. of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramol. complexity preserved during anal. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissocn. methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss tech. and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clin. and translational research.
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      Tian, Z.; Zhao, R.; Tolić, N.; Moore, R. J.; Stenoien, D. L.; Robinson, E. W.; Smith, R. D.; Paša-Tolić, L. Two-dimensional liquid chromatography system for online top-down mass spectrometry. Proteomics 2010, 10 (20), 3610– 3620,  DOI: 10.1002/pmic.201000367
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    24. 24
      Zhou, M.; Wu, S.; Stenoien, D. L.; Zhang, Z.; Connolly, L.; Freitag, M.; Pasa-Tolic, L. Profiling changes in histone post-translational modifications by top-down mass spectrometry. In Methods Mol. Biol. ; 2017; Vol. 1507, pp 153– 168.
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      Shaw, J. B.; Li, W.; Holden, D. D.; Zhang, Y.; Griep-Raming, J.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation. J. Am. Chem. Soc. 2013, 135 (34), 12646– 12651,  DOI: 10.1021/ja4029654
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      25
      Complete Protein Characterization Using Top-Down Mass Spectrometry and Ultraviolet Photodissociation
      Shaw, Jared B.; Li, Wenzong; Holden, Dustin D.; Zhang, Yan; Griep-Raming, Jens; Fellers, Ryan T.; Early, Bryan P.; Thomas, Paul M.; Kelleher, Neil L.; Brodbelt, Jennifer S.
      Journal of the American Chemical Society (2013), 135 (34), 12646-12651CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here the authors describe the implementation of 193 nm UV photodissocn. (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer's disease and in cancer pathogenesis. The 5 ns, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision- and electron-based dissocn. methods.
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    26. 26
      Cannon, J. R.; Holden, D. D.; Brodbelt, J. S. Hybridizing ultraviolet photodissociation with electron transfer dissociation for intact protein characterization. Anal. Chem. 2014, 86 (21), 10970– 10977,  DOI: 10.1021/ac5036082
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      26
      Hybridizing Ultraviolet Photodissociation with Electron Transfer Dissociation for Intact Protein Characterization
      Cannon, Joe R.; Holden, Dustin D.; Brodbelt, Jennifer S.
      Analytical Chemistry (Washington, DC, United States) (2014), 86 (21), 10970-10977CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      We report a hybrid fragmentation method involving electron transfer dissocn. (ETD) combined with UV photodissocn. (UVPD) at 193 nm for anal. of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the no. of identified c- and z-type ions obsd. when compared to UVPD or ETD alone, as well as generating a more balanced distribution of a/x, b/y, and c/z ion types. Addnl., the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissocn. (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total no. of fragment ions in comparison to the analogous MS3 format in which ETD and UVPD were undertaken in sep. segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liq. chromatog. timescale characterization was demonstrated for intact ribosomal proteins.
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    27. 27
      Cleland, T. P.; DeHart, C. J.; Fellers, R. T.; Vannispen, A. J.; Greer, J. B.; LeDuc, R. D.; Parker, W. R.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer. J. Proteome Res. 2017, 16 (5), 2072– 2079,  DOI: 10.1021/acs.jproteome.7b00043
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      27
      High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer
      Cleland, Timothy P.; DeHart, Caroline J.; Fellers, Ryan T.; VanNispen, Alexandra J.; Greer, Joseph B.; LeDuc, Richard D.; Parker, W. Ryan; Thomas, Paul M.; Kelleher, Neil L.; Brodbelt, Jennifer S.
      Journal of Proteome Research (2017), 16 (5), 2072-2079CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The anal. of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernible by anal. of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. UV photodissocn. (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatog. time scale. However, use of UVPD for high-throughput top-down anal. to date has been limited by bioinformatics. Here the authors detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher energy collisional dissocn. (HCD) in a comparative anal. of HeLa whole-cell lysate by qual. top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD data sets, with 68% of proteoforms having C scores >40 for UVPD and 63% for HCD. The av. sequence coverage (28 ± 20% for UVPD vs. 17 ± 8% for HCD, p < 0.0001) is higher for UVPD than HCD and with a trend toward improvement in q value for the UVPD data set. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtVCmu7w%253D&md5=7c377e118e8eae23532cbe620bebc197
    28. 28
      Tian, Z.; Tolić, N.; Zhao, R.; Moore, R. J.; Hengel, S. M.; Robinson, E. W.; Stenoien, D. L.; Wu, S.; Smith, R. D.; Paša-Tolić, L. Enhanced top-down characterization of histone post-translational modifications. Genome Biol. 2012, 13 (10), R86,  DOI: 10.1186/gb-2012-13-10-r86
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      28
      Enhanced top-down characterization of histone post-translational modifications
      Tian, Zhixin; Tolic, Nikola; Zhao, Rui; Moore, Ronald J.; Hengel, Shawna M.; Robinson, Errol W.; Stenoien, David L.; Wu, Si; Smith, Richard D.; Pasa-Tolic, Ljiljana
      Genome Biology (2012), 13 (), R86CODEN: GNBLFW; ISSN:1474-760X. (BioMed Central Ltd.)
      Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liq. chromatog.-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS anal. of 7.5 μg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVKjtrjP&md5=9f4120bb29c95543d76c1c7e608ef437
    29. 29
      Fort, K. L.; Dyachenko, A.; Potel, C. M.; Corradini, E.; Marino, F.; Barendregt, A.; Makarov, A. A.; Scheltema, R. A.; Heck, A. J. R. Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics. Anal. Chem. 2016, 88 (4), 2303– 2310,  DOI: 10.1021/acs.analchem.5b04162
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      29
      Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics
      Fort, Kyle L.; Dyachenko, Andrey; Potel, Clement M.; Corradini, Eleonora; Marino, Fabio; Barendregt, Arjan; Makarov, Alexander A.; Scheltema, Richard A.; Heck, Albert J. R.
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (4), 2303-2310CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissocn. (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biol. questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with UV photodissocn. (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liq. chromatog. (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amts. of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the anal. of post-translational modifications and is a powerful and complementary addn. to the proteomic toolbox.
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    30. 30
      Park, J.; Piehowski, P. D.; Wilkins, C.; Zhou, M.; Mendoza, J.; Fujimoto, G. M.; Gibbons, B. C.; Shaw, J. B.; Shen, Y.; Shukla, A. K. Informed-Proteomics: Open-source software package for top-down proteomics. Nat. Methods 2017, 14 (9), 909– 914,  DOI: 10.1038/nmeth.4388
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      30
      Informed-Proteomics: open-source software package for top-down proteomics
      Park, Jungkap; Piehowski, Paul D.; Wilkins, Christopher; Zhou, Mowei; Mendoza, Joshua; Fujimoto, Grant M.; Gibbons, Bryson C.; Shaw, Jared B.; Shen, Yufeng; Shukla, Anil K.; Moore, Ronald J.; Liu, Tao; Petyuk, Vladislav A.; Tolic, Nikola; Pasa-Tolic, Ljiljana; Smith, Richard D.; Payne, Samuel H.; Kim, Sangtae
      Nature Methods (2017), 14 (9), 909-914CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Top-down proteomics, the anal. of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liq. chromatog.-tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed. Here we present Informed-Proteomics, an open-source software suite for top-down proteomics anal. that consists of an LC-MS feature-finding algorithm, a database search algorithm, and an interactive results viewer. We compare our tool with several other popular tools using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up anal.
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    31. 31
      Leduc, R. D.; Fellers, R. T.; Early, B. P.; Greer, J. B.; Thomas, P. M.; Kelleher, N. L. The C-Score: A bayesian framework to sharply improve proteoform scoring in high-throughput top down proteomics. J. Proteome Res. 2014, 13 (7), 3231– 3240,  DOI: 10.1021/pr401277r
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      31
      The C-Score: A Bayesian Framework to Sharply Improve Proteoform Scoring in High-Throughput Top Down Proteomics
      LeDuc, Richard D.; Fellers, Ryan T.; Early, Bryan P.; Greer, Joseph B.; Thomas, Paul M.; Kelleher, Neil L.
      Journal of Proteome Research (2014), 13 (7), 3231-3240CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The automated processing of data generated by top down proteomics would benefit from improved scoring for protein identification and characterization of highly related protein forms (proteoforms). Here the authors propose the "C-score" (short for Characterization Score), a Bayesian approach to the proteoform identification and characterization problem, implemented within a framework to allow the infusion of expert knowledge into generative models that take advantage of known properties of proteins and top down anal. systems (e.g., fragmentation propensities, "off-by-1 Da" discontinuous errors, and intelligent weighting for site-specific modifications). The performance of the scoring system based on the initial generative models was compared to the current probability-based scoring system used within both ProSightPC and ProSightPTM on a manually curated set of 295 human proteoforms. The current implementation of the C-score framework generated a marked improvement over the existing scoring system as measured by the area under the curve on the resulting ROC chart (AUC of 0.99 vs. 0.78).
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      Lindner, H.; Sarg, B.; Meraner, C.; Helliger, W. Separation of acetylated core histones by hydrophilic-interaction liquid chromatography. J. Chromatogr. A 1996, 743 (1), 137– 144,  DOI: 10.1016/0021-9673(96)00131-8
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      Separation of acetylated core histones by hydrophilic-interaction liquid chromatography
      Lindner, Herbert; Sarg, Bettina; Meraner, Christoph; Helliger, Wilfried
      Journal of Chromatography A (1996), 743 (1), 137-144CODEN: JCRAEY; ISSN:0021-9673. (Elsevier)
      Hydrophilic-interaction liq. chromatog. (HILIC) has recently been introduced as a highly efficient chromatog. technique for the sepn. of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the sepn. of posttranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, vol./vol.) at pH 3.0. In combination with reversed-phase-high performance liq. chromatog. (RP-HPLC) we have successfully sepd. various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the anal. and isolation of modified core histone variants HILIC provides a new and important alternative to traditional sepn. techniques and will be useful in studying the biol. function of histone acetylation.
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      Lindner, H.; Sarg, B.; Helliger, W. Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones. J. Chromatogr. A 1997, 782 (1), 55– 62,  DOI: 10.1016/S0021-9673(97)00468-8
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      Benevento, M.; Tonge, P. D.; Puri, M. C.; Nagy, A.; Heck, A. J. R.; Munoz, J. Fluctuations in histone H4 isoforms during cellular reprogramming monitored by middle-down proteomics. Proteomics 2015, 15 (18), 3219– 3231,  DOI: 10.1002/pmic.201500031
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      Fluctuations in histone H4 isoforms during cellular reprogramming monitored by middle-down proteomics
      Benevento, Marco; Tonge, Peter D.; Puri, Mira C.; Nagy, Andras; Heck, Albert J. R.; Munoz, Javier
      Proteomics (2015), 15 (18), 3219-3231CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somatic cells into an embryonic-like pluripotent state. Post-translational modifications of histones play an important role in this process. Previously, we found by ChIP-seq widespread changes of specific histone H3 marks in two divergent reprogramming routes leading to alternative pluripotent sates . Here, using an unbiased middle-down proteomics approach we have identified 72 unique isoforms of histone H4 and quantified 56 of them in the same set of samples. We found substantial differences between somatic and late-phase reprogramming cells. Also, ESCs and iPSCs displayed higher levels of H4 acetylation and tri-methylation concomitantly with lower levels of mono- and di-methylation when compared to cells undergoing reprogramming. Our data shows that the epigenetic remodeling induced by the reprogramming process goes beyond histone H3 and reveals the importance of H4 modifications as well. The presented data is a valuable resource to study the epigenetic mechanisms involved in the acquisition of induced pluripotency. All MS data have been deposited in the ProteomeXchange with identifier PXD002062.
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      Young, N. L.; DiMaggio, P. a.; Plazas-Mayorca, M. D.; Baliban, R. C.; Floudas, C. A.; Garcia, B. A. High throughput characterization of combinatorial histone codes. Mol. Cell. Proteomics 2009, 8 (10), 2266– 2284,  DOI: 10.1074/mcp.M900238-MCP200
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      High throughput characterization of combinatorial histone codes
      Young, Nicolas L.; Di Maggio, Peter A.; Plazas-Mayorca, Mariana D.; Baliban, Richard C.; Floudas, Christodoulos A.; Garcia, Benjamin A.
      Molecular and Cellular Proteomics (2009), 8 (10), 2266-2284CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)
      We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liq. chromatog. directly coupled to electron transfer dissocn. (ETD) mass spectrometry for the automated online high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resoln. mass spectrometer, displays an improvement over existing methods with an ∼100-fold redn. in sample requirements and anal. time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h anal. The large N-terminal histone peptides are eluted by the pH and org. solvent weak cation exchange-hydrophilic interaction liq. chromatog. gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric tri-Me and acetyl modifications are resolved chromatog. and confidently distinguished by the synthesis of mass spectrometric and chromatog. information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the anal. of highly modified peptides. We find this methodol. very useful for chromatog. sepn. of isomeric species that cannot be sepd. well by any other chromatog. means, leading to less complicated tandem mass spectra. The improved sepn. and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3.2 forms and 70 H4 forms, including forms not yet detected in human cells, such as the remarkably highly modified histone H3.2 K4me3K9acK14acK18acK23acK27acK36me3. Such detail provided by our proteomics platform will be essential for detg. how histone modifications occur and act in combination to propagate the histone code during transcriptional events and could greatly enable sequencing of the histone component of human epigenomes.
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    36. 36
      Jung, H. R.; Sidoli, S.; Haldbo, S.; Sprenger, R. R.; Schwämmle, V.; Pasini, D.; Helin, K.; Jensen, O. N. Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS. Anal. Chem. 2013, 85 (17), 8232– 8239,  DOI: 10.1021/ac401299w
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      Precision Mapping of Coexisting Modifications in Histone H3 Tails from Embryonic Stem Cells by ETD-MS/MS
      Jung, Hye Ryung; Sidoli, Simone; Haldbo, Simon; Sprenger, Richard R.; Schwammle, Veit; Pasini, Diego; Helin, Kristian; Jensen, Ole N.
      Analytical Chemistry (Washington, DC, United States) (2013), 85 (17), 8232-8239CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, the authors report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone H3 N-terminal tail peptides (amino acids 1-50, 5-6 kDa) were sepd. by optimized weak cation exchange/hydrophilic interaction liq. chromatog. (WCX/HILIC) and sequenced online by electron transfer dissocn. (ETD) tandem mass spectrometry (MS/MS). High mass accuracy and near complete sequence coverage allowed unambiguous mapping of the major histone marks and discrimination between isobaric and nearly isobaric PTMs such as trimethylation and acetylation. Hierarchical data anal. identified H3K27me2-H3K36me2 as the most frequently obsd. PTMs in H3. Modifications at H3 residues K27 and K36 often coexist with the abundant mark K23ac, and the authors identified two frequently occurring quadruplet marks K9me1K23acK27me2K36me2 and K9me3K23acK27me2K36me, which might indicate a role in crosstalk. Co-occurrence frequency anal. revealed also an interplay between methylations of K9, K27, and K36, suggesting interdependence between histone methylation marks. The authors hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells.
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    37. 37
      Mizzen, C. A.; Alpert, A. J.; Lévesque, L.; Kruck, T. P. A.; McLachlan, D. R. Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography. J. Chromatogr., Biomed. Appl. 2000, 744 (1), 33– 46,  DOI: 10.1016/S0378-4347(00)00210-3
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      Resolution of allelic and non-allelic variants of histone H1 by cation-exchange-hydrophilic-interaction chromatography
      Mizzen, C. A.; Alpert, A. J.; Levesque, L.; Kruck, T. P. A.; McLachlan, D. R.
      Journal of Chromatography B: Biomedical Sciences and Applications (2000), 744 (1), 33-46CODEN: JCBBEP; ISSN:0378-4347. (Elsevier Science B.V.)
      A mixed-mode high-performance liq. chromatog. (HPLC) method that resolves the six known non-allelic variants of chicken erythrocyte histone H1 is described. Common, but previously unknown, allelic variants of H1 that co-migrate in PAGE are also resolved. The resoln. of H1 variants achieved by this method should be useful in detg. the functional significance of H1 sequence heterogeneity and in analyses of post-translational modification of H1. Furthermore, the principles behind the sepn. should be applicable to analyses of polymorphism in other proteins.
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      Papazyan, R.; Taverna, S. D. Separation and Purification of Multiply Acetylated Proteins Using Cation-Exchange Chromatography. Methods Mol. Biol. 2013, 981, 103– 113,  DOI: 10.1007/978-1-62703-305-3_8
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      Separation and purification of multiply acetylated proteins using cation-exchange chromatography
      Papazyan, Romeo; Taverna, Sean D.
      Methods in Molecular Biology (New York, NY, United States) (2013), 981 (Protein Acetylation), 103-113CODEN: MMBIED; ISSN:1064-3745. (Springer)
      High-performance liq. chromatog. (HPLC) is extremely useful for the study of proteins and the characterization of their posttranslational modifications. Here we describe a method that utilizes cation-exchange HPLC to sep. multiply acetylated histone H3 species on the basis of their charge and hydrophilicity. This high-resoln. method allows for the sepn. of histone H3 species that differ by as few as one acetyl group, and is compatible with subsequent anal. by a variety of techniques, including mass spectrometry and western blotting.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1Kku7zF&md5=ae7dc2f99b5dd62a0afec696165127c2
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      Lindner, H. H. Analysis of histones, histone variants, and their post-translationally modified forms. Electrophoresis 2008, 29 (12), 2516– 2532,  DOI: 10.1002/elps.200800094
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      Analysis of histones, histone variants, and their post-translationally modified forms
      Lindner, Herbert H.
      Electrophoresis (2008), 29 (12), 2516-2532CODEN: ELCTDN; ISSN:0173-0835. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. For many years, histones were considered passive structural components of eukaryotic chromatin. Meanwhile it has been proven that histones also participate in gene regulation and repression via post-translational modification. The multitude of these post-translational modifications and the existence of numerous histone variants require particular sepn. strategies for their anal., a prerequisite for studying biol. processes. The most widely utilized techniques for the sepn. of histones, namely PAGE, HPCE, RP-HPLC, and hydrophilic Interaction LC, are reviewed here. Problems inherent to the anal. of histones owing to their unique phys. and chem. properties along with advantages and shortcomings of particular methods are discussed.
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      Zheng, Y.; Fornelli, L.; Compton, P. D.; Sharma, S.; Canterbury, J.; Mullen, C.; Zabrouskov, V.; Fellers, R. T.; Thomas, P. M.; Licht, J. D. Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression. Mol. Cell. Proteomics 2016, 16 (18), 1– 40,  DOI: 10.1074/mcp.M115.053819
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      Analyses of histone H3 from 10 rat tissues using a Middle Down proteomics platform revealed tissue-specific differences in their expression and global PTM abundance. ESI/FTMS with electron capture dissocn. showed that, in general, these proteins were hypomodified in heart, liver and testes. H3.3 was hypermodified compared to H3.2 in some, but not all tissues. In addn., a novel rat testes-specific H3 protein was identified with this approach.
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      Zhou, M.; Paša-Tolić, L.; Stenoien, D. L. Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry. J. Proteome Res. 2017, 16 (2), 599– 608,  DOI: 10.1021/acs.jproteome.6b00694
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      Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry
      Zhou, Mowei; Pasa-Tolic, Ljiljana; Stenoien, David L.
      Journal of Proteome Research (2017), 16 (2), 599-608CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biol. and their roles in disease development have been the subject of intense study. Since multiple PTMs along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone anal. primarily because of deficiencies in sepn./resolving power and effective identification algorithms. Here, the authors used state of the art mass spectrometry and a bioinformatics workflow for targeted data anal. and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.
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      Quant. proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and abs. quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >104 using a strictly Top Down mass spectrometric approach coupled with two dimensions of liq. chromatog. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quant. analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quant. anal. This work represents the most comprehensive anal. of histone H4 forms present in human cells reported to date.
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      Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae
      Vonk, Rudy J.; Gargano, Andrea F. G.; Davydova, Ekaterina; Dekker, Henk L.; Eeltink, Sebastiaan; de Koning, Leo J.; Schoenmakers, Peter J.
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      Stationary-phase-assisted modulation was used to overcome one of the limitations of contemporary comprehensive two-dimensional liq. chromatog., which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high diln. of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. Stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diams. without loss in system performance. In the current setup, a 0.30 mm internal diam. first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diam. second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resoln. mass spectrometer and applied for the sepn. of the tryptic peptides of a six-protein mixt. and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, ∼20000 MS/MS spectra were generated within 24 h anal. time, resulting in the identification of 701 proteins.
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      Yuan, Z. F.; Sidoli, S.; Marchione, D. M.; Simithy, J.; Janssen, K. A.; Szurgot, M. R.; Garcia, B. A. EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data. J. Proteome Res. 2018, 17 (7), 2533– 2541,  DOI: 10.1021/acs.jproteome.8b00133
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      EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data
      Yuan, Zuo-Fei; Sidoli, Simone; Marchione, Dylan M.; Simithy, Johayra; Janssen, Kevin A.; Szurgot, Mary R.; Garcia, Benjamin A.
      Journal of Proteome Research (2018), 17 (7), 2533-2541CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Epigenetics has become a fundamental scientific discipline with various implications for biol. and medicine. Epigenetic marks, mostly DNA methylation and histone posttranslational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here the authors present EpiProfile 2.0, an extended version of the 2015 software (v1.0), for accurate quantification of histone peptides based on liq. chromatog.-tandem mass spectrometry (LC-MS/MS) anal. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extd. ion chromatog. to accurately det. the chromatog. profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, the authors anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biol. and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family.
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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.8b00458.

    • Gradient programming of the 2DLC (Supporting Material S1, Table S1), a schematic illustration of the histone identification workflow (Supporting Material S2, Figure S1). Additional data on 1DLC and 2DLC separations are reported in the Supporting Material S3. This include details of the separation families by 1DLC WCX-HILIC (Figure S2), 1DLC RPLC (Figure S3), 2DLC WCX-HILIC/a×m/RPLC (Figure S4), the TIC of a single modulation of the 2DLC separation (Figure S5), and the analysis of the distribution of neutral masses detected for each histone family (Figure S6). The list of families of proteins identified using WCX-HILIC/a×m/RPLC UVPD-HRMS (Prosight PC 4.0) is collected in Supporting Material S4, Table S2. The results from the triplicate analysis of HeLa Histones using the WCX-HILIC/a×m/RPLC UVPD-HRMS platform are in the Supporting Material S5, Figure S7. In Supporting Materials S6 and S7 are reported examples of proteoform IDs obtained with our analysis workflow using respectively the informed proteomics and Prosight PC data analysis toolkit. Figure S8 shows a detail of Figure 2 reporting the monoisotopic mass area of H3 histones and assignment of H3.2 proteoforms using the informed proteomics workflow and Figure S9–S14 examples of H3.2 proteoform IDs. Figure S15 shows a detail of Figure 2 reporting the monoisotopic mass area of H4 histones and assignment of H4 proteoforms using the informed proteomics workflow and Figure S16–S19 examples of H4 proteoform IDs. Examples of sequence coverage of protein identifications using Prosight PC 4.0 are collected in Supporting Material S7 (PDF)

    • Excel file reporting the output of the Prosight PC 4.0 searches summarized in Table S3 (list of proteoform identified by Prosight PC 4.0 using a restricted database) (XLSX)

    • Excel file reporting the output of the Prosight PC 4.0 searches summarized in Table S4 (list of proteoforms identified using Prosight PC 4.0 using the Human Proteome database and filtered by C score) (XLSX)


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