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Abstract
Multiple reaction monitoring tandem mass spectrometry becomes an important strategy for measuring protein targets in complex biomatrixes. Active chemical modification of peptides like phenylthiocarbamoylation has unique potential for improving the measurement. This potential is enabled by active participation of a modifying group in site-preferential dissociation of modified peptides, which produces certain fragment ions at very high yields and in a sequence-independent manner. In this work, a novel combination of energy-resolved mass spectrometry with substituent effect investigation is used to analyze important factors that control the specificity of the site-preferential dissociation of phenylthiocarbamoyl peptides. On the basis of the linear correlation between collision energy and the Hammett constant as well as computational studies, it is found that the initial enhanced capture of a mobile proton and the subsequent, site-directed intramolecular proton transfer are important to the high yields (∼70−90%) for producing two types of fragment ions of phenylthiocarbamoyl peptides: the modified b1 ion and the complementary yn−1 ion. This understanding will help the design of new modification reagents. When integrated with the throughput and the signal-enhancing potential of peptide modification, active chemical modification of peptides will significantly advance mass spectrometry-based, targeted proteome analysis.
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After a decade of fast advance in proteomics, one important task for protein measurements is to efficiently monitor and quantify target proteins in complex biomatrixes. These targets are accumulated from conventional biochemical and biological investigations as well as from discovery proteome profiling experiments. Targeting a specific set of proteins for focused mass spectrometry is an increasingly attractive strategy for proteome-wide quantitative investigation of biological pathways and cellular signal transduction, as well as for monitoring of human disease biomarkers. (1-8) Multiple reaction monitoring (MRM) tandem mass spectrometry (MS/MS) and its variations are often the methods of choice, which use signature peptides as surrogate markers for their precursor proteins. (1-8) MRM MS/MS uses a particular gas-phase reaction(s) for measuring a peptide, and thus its detection limit depends on both the signal intensity of the precursor and that of a selected fragment ion(s) of the peptide. An intact peptide that has a strong signal, however, does not necessarily produce fragments at high yields due to multiple fragment pathways for activated peptide ions. (9-11) Amino acid composition and sequence needed for efficient generation of intact peptide ions and for effective production of fragment ions are not necessarily the same. A compromise between signal intensities for precursor and fragment ions has often been made during signature peptide selection in MRM MS/MS experimental design, which results in less-than-optimal detection limits. Furthermore, signature peptides for MRM measurements, (5) even those with bonds capable of biased fragmentation, (12, 13) typically produce the corresponding fragment ions at yields less than 10%. These challenges in selecting signature peptides can be significant. For instance, small target proteins can only generate a few peptides without bonds of preferential fragmentation potential.
Active chemical modification (14) of peptides provides a promising, universal solution for generating fragmentation ions at high yields for enhancing the detection and quantitation limits for MRM MS/MS. This is a chemical modification approach; (15) a modifying group actively participates in controlling the specificity of gas-phase peptide bond dissociation, which produces particular fragment ions at very high yields. Importantly, the bond dissociation has little dependence on peptide amino acid composition and sequence and this eliminates the need of searching for signature peptides with biased fragmentation potential. This independency on peptide sequence for high yield fragment ion generation releases the fragment ion signal as the limiting factor for the detection limit for MRM MS/MS analysis. Active chemical modification has a well-known example, phenylthiocarbamoylation, although its applications in MRM MS/MS of peptides have not been broadly exploited. Phenylthiocarbamoyl peptides provide a system for examining important factors that need to be considered in design of new reagents for active chemical modification of peptides for MRM measurements. Upon random gas-phase collision, (11) the fragmentation of N-terminally modified phenylthiocarbamoyl peptides can be highly specific to the first N-terminal peptide bond. (14, 16-22) This specificity has been solely attributed to the high nucleophilicity of the thioxo sulfur atom, relative to amide oxygen atoms on the same peptide. (16, 17, 23) The role of the high gas-phase basicity of the thioxo sulfur (24) in the specificity control has not been examined.
Activation of amide bonds by a mobile proton facilitates gas-phase collision-induced dissociation of peptides, an important process for peptide mass spectrometry. (9-11) In analogy to the mechanism for the preferential cleavage at the C-side amide bond of histidine, (25) a triad mechanism (Scheme 1) is proposed to test if site-directed protonation of the first amide bond is essential to the selectivity control for dissociation of phenylthiocarbamoyl peptides. This mechanism has three sequential steps: (A) enhanced capture of a mobile proton (9) by the basic thioxo sulfur (24) that acts as an initial “proton antenna” (1 to 2); (B) intramolecular transfer of the trapped proton to the adjacent peptide bond (2 to 3), which results in the site-specific activation of the first N-terminal peptide bond; (C) nucleophilic attack of the activated amide group by the deprotonated thioxo sulfur atom (from 3 to b1 and yn−1). (16, 17) Herein, we report a novel use of energy-resolved mass spectrometry for detecting the site-directed localization of a mobile proton in ring-substituted phenylthiocarbamoyl peptides.
Scheme 1
Peptide NSILTETLHR of cystic fibrosis transmembrane conductance regulator protein was modified by reacting it with various ring-substituted phenylisothiocyanates (Figure 1a) following a reported procedure. (22) Tandem mass spectra of the modified peptides showed the preferential cleavage of the first N-terminal amide bond (Figure S1 in the Supporting Information). The modified b1 and the complementary y9 ions (Scheme 1) for the 4-iodophenylthiocarbamoyl peptide were dominantly observed (Figure S1 in the Supporting Information) and their maximum MRM MS/MS yields were measured to be 89% and 67%, respectively, by energy-resolved mass spectrometry (Figure 2) on a triple quadrupole tandem mass spectrometer (ABI 4000 QTrap). The yields were calculated based on the percentages of the maximal relative intensities for the MRM transitions of [M + 2H]2+ → b, (y9 or y7) over [M + 2H]2+ → [M + 2H]2+, respectively. High yields for producing the modified b1 ion and the complementary yn−1 from phenylthiocarbamoyl peptides are beneficial to MRM measurements of target peptides in protein digests (Figure S2 in the Supporting Information).
Figure 1
Figure 1. Substituent effect on gas-phase dissociation of ring-substituted phenylthiocarbamoyl peptides. The structure (a) shows the modified peptides with two protons, one at the cleavable peptide bond and the other at the C-terminal arginine; only the first two amino acid residues are illustrated. Part b is an overlay of multiple collision energy profiles for producing the y9 ion from a modified peptide with the maximal collision energy (CEmax) located.
Figure 2
Figure 2. Energy-resolved mass spectrometry comparison of yields for fragment ions generated from a 4-iodophenylthiocarbamoyl peptide with a sequence of NSILTETLHR. Ion transitions include [M + 2H]2+/[M + 2H]2+ (maximum yield, 100%; normalization standard), [M + 2H]2+/b1* (maximum yield, 89%), [M + 2H]2+/y9 (maximum yield, 67%) and [M + 2H]2+/y7 (maximum yield, 5%).
Energy-resolved dissociation of all of the ring-substituted phenylthiocarbamoyl peptides was monitored. The dependence of signal intensity on the collision energy offset voltage (CE) for the collision cell was monitored for the singly charged y9 ion (SILTETLHR, theoretical m/z 1069.6005), which is common to all of the modified peptides. Replicated experiments (12−101 times on multiple days and multiple sample preparations) were conducted for each modified peptide. The CE profiles for the y9 ion for a particular modified peptide were first normalized, separately, to their own approximate average maximum CE values [obtained from the Gaussian fitting of the profiles using Origin 8 (OriginLab, MA)]. All of the normalized CE profiles for the modified peptide were then combined. A defined range [from the top 50% data for the lower CE end (left) of the profile and the top 80% data for the higher CE end (right)] of data in the combined CE profile, as in Figure S3 in the Supporting Information, were fitted with an asymmetric double sigmoid function using Origin 8. The CE value corresponding to the maximal ion intensity was designated as CEmax for the peptide (Figure 1b and Figure S3 in the Supporting Information). Calculated CEmax had high precision; when the error for a CEmax was smaller than 0.1 V (the step size for CE increase for the collision cell in the energy-resolved experiments), 0.1 V was used as the experimental error for the laboratory CEmax values.
Hammett constants for substituents (26) on the phenyl ring were correlated with laboratory CEmax values for the y9 ion (Figure 3). A linear relationship was established based on all meta substituents (Y or Z substituents, Figure 1a), giving a positive slope of 5.1(±0.8). Substituents 4-dimethylamino (a, Figure 3) and 4-diethylamino (b, Figure 3) had significant positive deviations from the linear regression. The CE profile for the y9 ion is the competitive sum of the CE dependencies on the ion generation and on further dissociation of the ion. Values for CEmax are used as relative measurements, by the first approximation, of the dissociation of the first N-terminal amide bond. This approximation is based on the following considerations. First, fragments generated from competing dissociation pathways other than the first N-terminal amide dissociation are minimal; they are y92+ ion and a doubly charged peptide ion losing the modifying group (Figures S1 and S4 in the Supporting Information). Second, the origin of the y9 ion generation is attributed to the primary cleavage of the first N-terminal peptide bond, and the y9 ion is the common fragment for all of the modified peptides. Simpler model systems, however, are needed to further analyze contributing factors to the observed CEmax values, including differences in the peptide mass and vibrational degree of freedom.
Figure 3
Figure 3. Correlation of laboratory CEmax with the Hammett constant. Substituent a, 4-dimethylamino; b, 4-diethylamino; c, 4-methoxy; d, 4-ethoxy; e, 3,4-dimethoxy; f, 3,5-dimethyl; g, 3-methyl; h, unsubstituted; i, 3,4,5-trimethoxy; j, 3-methoxy; k, 3-methylthio; l, 4-iodo; m, 3-iodo; n, 3-bromo; o, 3-chloro; p, 3-methoxycarbamoyl; q, 4-methoxycarbonyl; r, 3-cyano; s, 4-cyano; t, 3-nitro; and u, 4-nitro. The circle labels are for para substituents: solid red for strong resonance electron-donating and open blue for resonance electron-withdrawing. The solid squares are for meta substituents.
The positive slope of the linear correlation for the y9 ion (Figure 3) supports the importance of the previously proposed, thioxo sulfur nucleophilicity in enhancing fragmentation of the first peptide bond in phenylthiocarbamoyl peptides (3). (16, 17) The presence of the less electron-withdrawing substituent (i.e., the decreased Hammett constant) makes the thioxo sulfur a stronger nucleophile for intramolecular attack of the successive peptide bond to form 4, which results in the decreased CEmax. The high nucleophilicity of thioxo sulfur has also been accounted for the preferential cleavage in thioxo peptides. (27)
Substituents 4-dimethylamino and 4-diethylamino, capable of strong electron-donating stabilization through resonance, were selected as the probes for differentiating the substituent effect on the intramolecular proton transfer (2 to 3, Scheme 1) from that on the nucleophilic attack (3 to 4). These two processes have opposite substituent effects. Electron-donating resonance groups prohibit the proton transfer, decrease the peptide bond cleavage, and increase CEmax. On the other hand, these para substituents enhance the thioxo sulfur nucleophilicity, facilitate the peptide bond cleavage, and decrease CEmax. It would be expected that if the only controlling factor for the site-specific amide bond cleavage were the sulfur nucleophilicity as previously proposed, (16, 17) then these resonance probing substituents would show negative deviations from the linear correlation.
In contrast, considerable positive deviations were observed for 4-dimethylamino and 4-diethylamino substituents (Figure 3) and they revealed the presence of a stabilized, protonated thiocarbamoyl group (Figure S5 in the Supporting Information) in 2b. This resonance stabilization prohibits the intramolecular proton transfer from 2 to 3. The reduced intramolecular proton transfer results in the decreased, site-directed activation of the first amide bond. However, it is important to note that the intramolecular proton transfer from 2 to 3 does not play a sole role in limiting the peptide bond dissociation. Otherwise, a negative slope for the correlation of the CEmax against the Hammett constant would be observed. Thus, combined observations of the positive slope for the correlation and positive deviations of the protonation probing substituents indicate that both 2 to 3 and 3 to 4 (Scheme 1) might be rate-limiting steps for the site-preferential dissociation of phenylthiocarbamoyl peptides. Simpler compounds than the modified peptides used could facilitate further examination of kinetics and mechanisms of gas-phase dissociation of phenylthiocarbamoyl peptides.
The high gas-phase basicity of thioxo sulfur (24) is proposed to be responsible for the initial mobile proton capture by the phenylthiocarbamoyl modifying group in 1 to form 2 (Scheme 1), in competition with other amide groups for the same mobile proton. The substituent effect on the initial sulfur protonation is also qualitatively observed in tandem mass spectra of the modified peptides (Figure S1 in the Supporting Information). Strong electron-withdrawing substituents like 4-nitro on the phenyl ring decrease the sulfur basicity (1) so that the sulfur atom becomes less competitive for the mobile proton to initiate the site-preferential fragmentation. As a result, dissociation products from other pathways increase (Figures S1 and S4 in the Supporting Information). In comparison, the oxygen atom in the modifying group of phenylcarbamoyl peptides is not basic enough to enhance capture of a mobile proton. Therefore, phenylcarbamoyl peptides undergo multiple peptide bond dissociation. (14, 22) Phenylcarbamoylation of peptides is thus referred to as passive chemical modification, in which the modifying group performs as an inert mass tag. (14)
The initial sulfur protonation and subsequent intramolecular proton transfer (1 to 2 to 3 in Scheme 1) are also supported by computational studies of a model compound 2-[3-(4-dimethylamino-phenyl)-ureido]-N-methylcarbamoylmethyl-acetamide. With the use of density functional theory (DFT) with hybrid functional B3LYP and basis set 6-31 g**, relative energies for protonation isomers of the model compound and the transition state (obtained via the linear synchronous transit method) for intramolecular proton transfer were calculated with the Jaguar program. Initial protonation of thioxo sulfur (defined as the zero of energy) is much favorable over that of the adjacent amide oxygen (19.9 kcal/mol, Figure S6 in the Supporting Information). Protonation of the dimethylamino nitrogen also requires higher energy (1.7 kcal/mol). The rotational isomer of the sulfur protonated molecule (0 kcal/mol) forms an intramolecular hydrogen bond with a slightly higher energy (0.5 kcal/mol, Figure S6 in the Supporting Information), which can then transfer the proton to the adjacent amide oxygen (0.3 kcal/mol) through a transition state with a low activation barrier (0.7 kcal/mol, Figure S7 in the Supporting Information).
Chemical manipulation of peptide fragment ion yields or gas-phase peptide dissociation is less explored than chemical enhancement of mass spectral signals for intact peptide ions. Modification of peptides for introducing basic group and/or increasing peptide hydrophobicity has been shown beneficial to enhancing MS signals for intact, modified peptides, (28, 29) which can improve MRM detection limits by increasing signals for precursor peptide ions. Chemical tuning of fragment ion yields for modified peptides requires an “active” involvement of a modifying group in gas-phase dissociation of a modified peptide. In other words, the modifying group participates in gas-phase dissociation of the modified peptide. Peptides modified by phenylthiocarbamoylation of the N-terminal amine with substituted phenylisothiocyanates provide a unique system for understanding principles governing such a chemical tuning of peptide dissociation. Preferential gas-phase cleavage of the first amino acid residue is also commonly observed in peptides modified with reductive alkylation, (30-32) another type of active chemical modification. (14)
In conclusion, this work timely contributes to the area of targeted mass spectrometry, especially MRM measurements, of protein biomarkers. It addresses an important but previously less appreciated issue for improving MRM measurements: how to increase the fragment ion yield. Fragment ion signals and precursor signals, together, define the MRM limits of detection and quantitation. Controllable peptide cleavage as the essence of active chemical modification provides a means to rationally improve the fragment ion yield. Important contributors to this improvement are identified based on a novel application of energy-resolved mass spectrometry. This understanding will help the design and development of new reagents for improved MRM measurements. The scope of active chemical modification can be expanded by integration with the throughput potential of peptide labeling, which enables mass spectrometric analysis of a common analyte in several samples, (33) and the signal-enhancing potential, which increases the mass spectrometric signals for peptides through chemical introduction of signal enhancing moieties. (28, 29) Significant advances in targeted proteome analysis using MRM MS/MS and novel modification reagents are thus foreseeable.
Supporting Information
Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgment
This work was supported by the University of Connecticut, the Cystic Fibrosis Foundation (Grant YAO07XX0), and Grant IGR-06-002-01 from the American Cancer Society. Fata H. Koudoro supported by the NSF REU program at the University of Connecticut did the preliminary studies. This work was in part presented at the 57th ASMS Conference on Mass Spectrometry and Allied Topics in 2009. The authors thank M. V. Stipdonk for sharing a 57th ASMS poster entitled “Effect of Ring Substituents on the Dissociation Behavior of Model, Benzoic Acid Terminated Peptide and Esters.”
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- 8Lange, V., Malmstrom, J. A., Didion, J., King, N. L., Johansson, B. P., Schafer, J., Rameseder, J., Wong, C. H., Deutsch, E. W., Brusniak, M. Y., Buhlmann, P., Bjorck, L., Domon, B. and Aebersold, R. Mol. Cell. Proteomics 2008, 7, 1489– 1500Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVSrs7vO&md5=23244501fc3a8219b4b4ed69b44c8210Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoringLange, Vinzenz; Malmstrom, Johan A.; Didion, John; King, Nichole L.; Johansson, Bjorn P.; Schafer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W.; Brusniak, Mi-Youn; Buhlmann, Peter; Bjorck, Lars; Domon, Bruno; Aebersold, RuediMolecular and Cellular Proteomics (2008), 7 (8), 1489-1500CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)In many studies, particularly in the field of systems biol., it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such expts. has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quant. approach by which predetd. protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quant. Anal. by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amts. of plasma. The resulting quant. protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.
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- 11Wells, J. M. and McLuckey, S. A. Methods Enzymol. 2005, 402, 148– 185Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XosFeruro%253D&md5=849f2e36d0a18d1ed3c716025970cba6Collision-induced dissociation (CID) of peptides and proteinsWells, J. Mitchell; McLuckey, Scott A.Methods in Enzymology (2005), 402 (Biological Mass Spectrometry), 148-185CODEN: MENZAU; ISSN:0076-6879. (Elsevier)A review. The most commonly used activation method in the tandem mass spectrometry (MS) of peptides and proteins is energetic collisions with a neutral target gas. The overall process of collisional activation followed by fragmentation of the ion is commonly referred to as collision-induced dissocn. (CID). The structural information that results from CID of a peptide or protein ion is highly dependent on the conditions used to effect CID. These include, for example, the relative translational energy of the ion and target, the nature of the target, the no. of collisions that is likely to take place, and the observation window of the app. This chapter summarizes the key exptl. parameters in the CID of peptide and protein ions, as well as the conditions that tend to prevail in the most commonly employed tandem mass spectrometers.
- 12Huang, Y., Triscari, J. M., Tseng, G. C., Pasa-Tolic, L., Lipton, M. S., Smith, R. D. and Wysocki, V. H. Anal. Chem. 2005, 77, 5800– 5813Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXotVSlsbk%253D&md5=b7a412931e70727b82c21993a5c58423Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation PatternsHuang, Yingying; Triscari, Joseph M.; Tseng, George C.; Pasa-Tolic, Ljiljana; Lipton, Mary S.; Smith, Richard D.; Wysocki, Vicki H.Analytical Chemistry (2005), 77 (18), 5800-5813CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Data mining was performed on 28,330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chem. interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissocn. patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliph. residues (Ile, Val, Leu) are obsd. in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are obsd. in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns obsd. expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities.
- 13Huang, Y., Tseng, G. C., Yuan, S., Pasa-Tolic, L., Lipton, M. S., Smith, R. D. and Wysocki, V. H. J. Proteome Res. 2008, 7, 70– 79Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlygsr%252FK&md5=e4c2967f5d64fdeea40fa9c9a6db85d2A Data-Mining Scheme for Identifying Peptide Structural Motifs Responsible for Different MS/MS Fragmentation Intensity PatternsHuang, Yingying; Tseng, George C.; Yuan, Shinsheng; Pasa-Tolic, Ljiljana; Lipton, Mary S.; Smith, Richard D.; Wysocki, Vicki H.Journal of Proteome Research (2008), 7 (1), 70-79CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Although tandem mass spectrometry (MS/MS) has become an integral part of proteomics, intensity patterns in MS/MS spectra are rarely weighted heavily in most widely used algorithms because they are not yet fully understood. Here a knowledge mining approach is demonstrated to discover fragmentation intensity patterns and elucidate the chem. factors behind such patterns. Fragmentation intensity information from 28 330 ion trap peptide MS/MS spectra of different charge states and sequences went through unsupervised clustering using a penalized K-means algorithm. Without any prior chem. assumptions, four clusters with distinctive fragmentation patterns were obtained. A decision tree was generated to investigate peptide sequence motif and charge state status that caused these fragmentation patterns. This data-mining scheme is generally applicable for any large data sets. It bypasses the common prior knowledge constraints and reports on the overall peptide fragmentation behavior. It improves the understanding of gas-phase peptide dissocn. and provides a foundation for new or improved protein identification algorithms.
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- 26Hansch, C., Leo, A. and Taft, R. W. Chem. Rev. 1991, 91, 165– 195Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhs1ehsLo%253D&md5=9fc814cd57c47680a5213f3438037800A survey of Hammett substituent constants and resonance and field parametersHansch, Corwin; Leo, A.; Taft, R. W.Chemical Reviews (Washington, DC, United States) (1991), 91 (2), 165-95CODEN: CHREAY; ISSN:0009-2665.Included in this review is an anal. of newer methods which can supplant this classic procedure for detn. of the title consts., 283 refs.
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- 29Williams, D. K., Jr., Meadows, C. W., Bori, I. D., Hawkridge, A. M., Comins, D. L. and Muddiman, D. C. J. Am. Chem. Soc. 2008, 130, 2122– 2123Google ScholarThere is no corresponding record for this reference.
- 30Hsu, J.-L., Huang, S.-Y., Shiea, J.-T., Huang, W.-Y. and Chen, S.-H. J. Proteome Res. 2005, 4, 101– 108Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFWqsr%252FK&md5=24d15f458030df9cb2df9df759ba9e95Beyond Quantitative Proteomics: Signal Enhancement of the a1 Ion as a Mass Tag for Peptide Sequencing Using Dimethyl LabelingHsu, Jue-Liang; Huang, Sheng-Yu; Shiea, Jen-Taie; Huang, Wen-Ying; Chen, Shu-HuiJournal of Proteome Research (2005), 4 (1), 101-108CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Stable isotope-based di-Me labeling that produces a di-Me labeled terminal amine or a monomethylated proline N-terminus by reductive methylation (Anal. Chem. 2003, 75, 6843-6852) was reported as a promising strategy for global quant. proteomics because of the simplicity of the process and its fast and complete reaction. This labeling strategy provides a signal enhancement for the produced a1 ions, which are usually hard to detect among most of the nonderivatized fragments. To assist peptide sequencing, in this study, the enhanced a1 ion produced under either collision induced dissocn. (CID) or post source decay (PSD) modes was further characterized and applied as a mass tag for fingerprinting the identity of N-terminal amino acid. On the basis of the anal. of std. peptides, tryptic digests of Hb and cell lysates, it was proved that such signal enhancement occurred to a1 ions derived from all 20 of the amino acids residues and this phenomenon was explained based the formation of stable quaternary immonium ions. Accurate detn. of a1 ions was shown to increase the chance for peptide de novo sequencing and also provided higher confidence in the scores obtained when identifying a protein through database searching. In addn., the a1 ion was further demonstrated to be used as a universal tag for precursor ion scan in a Q-TOF instrument, leading to a greater no. of peptide ions sequenced. Combined with the capability for differential quantitation, the stable isotope-based di-Me labeling increases the usefulness of the labeling method for MS-based proteomics.
- 31Fu, Q. and Li, L. Anal. Chem. 2005, 77, 7783– 7795Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFOis7fL&md5=599df751d27b143c9eef5ba30d9cae14De Novo Sequencing of Neuropeptides Using Reductive Isotopic Methylation and Investigation of ESI QTOF MS/MS Fragmentation Pattern of Neuropeptides with N-Terminal DimethylationFu, Qiang; Li, LingjunAnalytical Chemistry (2005), 77 (23), 7783-7795CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A stable-isotope di-Me labeling strategy was previously shown to be a useful tool for quant. proteomics. More recently, N-terminal di-Me labeling was also reported for peptide sequencing in combination with database searching. Here, the authors extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue ext. without any genomic information. The authors demonstrated several new sequencing applications of this method in addn. to the identification of the N-terminal residue using the enhanced a1 ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y(n-1) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also obsd. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of Nα, Nε-tetramethylated lysine and Nε-dimethylated lysine are explored.
- 32Locke, S. J., Leslie, A. D., Melanson, J. E. and Pinto, D. M. Rapid Commun. Mass Spectrom. 2006, 20, 1525– 1530Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVartb8%253D&md5=0e6a5927fa3fefbfcea78e1eca23b508Deviation from the mobile proton model in amino-modified peptides: implications for multiple reaction monitoring analysis of peptidesLocke, Steven J.; Leslie, Andrew D.; Melanson, Jeremy E.; Pinto, Devanand M.Rapid Communications in Mass Spectrometry (2006), 20 (10), 1525-1530CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)The study of peptide fragmentation is important to the understanding of chem. processes occurring in the gas phase and the more practical concern of peptide identification for proteomic anal. Using the mobile proton model as a framework, the authors explored the effect of amino-group modifications on peptide fragmentation. Three aldehydes were used to transform the primary amino groups on peptides into either a dimethylamino or a heterocyclic structure (five- or six-membered). The obsd. fragmentation patterns deviated strongly from those obsd. for the analogous underivatized peptides. In particular, the a1 ion was the base peak in most tandem mass spectra of the derivatized peptides. It was obsd. that the a1 ion intensity depends strongly on the N-terminal amino acid, with tyrosine and phenylalanine having the strongest enhancement. Despite the change in fragmentation patterns of the derivatized peptides, they still provided high-quality tandem mass spectra that, in many cases, are more amenable to database searching than the spectra of underivatized peptides. In addn., the reliable presence of the a1 ion facilitated rapid quant. measurements using the multiple reaction monitoring approach.
- 33Desouza, L. V., Taylor, A. M., Li, W., Minkoff, M. S., Romaschin, A. D., Colgan, T. J. and Siu, K. W. J. Proteome Res. 2008, 7, 3525– 3534Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXosFKmsbg%253D&md5=8f1b2405f6537fe6f41269fb28c560b2Multiple Reaction Monitoring of mTRAQ-Labeled Peptides Enables Absolute Quantification of Endogenous Levels of a Potential Cancer Marker in Cancerous and Normal Endometrial TissuesDe Souza, Leroi V.; Taylor, Adrian M.; Li, Wei; Minkoff, Marjorie S.; Romaschin, Alexander D.; Colgan, Terence J.; Siu, K. W. MichaelJournal of Proteome Research (2008), 7 (8), 3525-3534CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)While iTRAQ analyses have proved invaluable for the discovery of potential cancer markers, two outstanding issues that remained were its ineffectiveness to consistently detect specific proteins of interest in a complex sample and to det. the abs. abundance of those proteins. These have been addressed by availability of the mTRAQ reagents (Applied Biosystems, Inc., Foster City, CA) a nonisobaric variant of iTRAQ. We have applied this newly emerging technique to quantify one of our potential markers for endometrial cancer, viz. pyruvate kinase M1/M2. The mTRAQ methodol. relies on multiple reaction monitoring (MRM) to target tryptic peptides from the protein of interest, thus, ensuring maximal opportunity for detection, while the nonisobaric tags enable specific quantification of each version of the labeled peptides through unique MRM transitions conferred by the labels. Known amts. of synthetic peptides tagged with one of the two available mTRAQ labels, when used as quantification stds. in a mixt. with the oppositely labeled tryptically digested sample, permit detn. of the abs. amts. of the corresponding protein in the sample. The ability to label the sample and ref. peptides with either one of the two possible combinations is an inherent advantage of this method, as it provides a means for verification of the reported ratios. In this study, we detd. that the amt. of pyruvate kinase present in the homogenate from a biopsied EmCa tissue sample was 85 nmol/g of total proteins, while the equiv. concn. in the nonmalignant controls was 21-26 nmol/g of total proteins. This approx. 4-fold higher amt. of pyruvate kinase in the cancer sample was further confirmed not only by a direct comparison between the cancer sample and one of the nonmalignant controls, but also independently by an enzyme-linked immunosorbant assay (ELISA). Addnl., the 4-fold higher level of pyruvate kinase amt. in the cancer homogenate reported in this study is considerably higher than the 2-fold higher ratio reported across 20 cancer samples in the discovery phase with the iTRAQ technique, suggesting that there exists a possibility that the dynamic range of ratios detd. by the iTRAQ technique may have been compressed.
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Scheme 1
Figure 1
Figure 1. Substituent effect on gas-phase dissociation of ring-substituted phenylthiocarbamoyl peptides. The structure (a) shows the modified peptides with two protons, one at the cleavable peptide bond and the other at the C-terminal arginine; only the first two amino acid residues are illustrated. Part b is an overlay of multiple collision energy profiles for producing the y9 ion from a modified peptide with the maximal collision energy (CEmax) located.
Figure 2
Figure 2. Energy-resolved mass spectrometry comparison of yields for fragment ions generated from a 4-iodophenylthiocarbamoyl peptide with a sequence of NSILTETLHR. Ion transitions include [M + 2H]2+/[M + 2H]2+ (maximum yield, 100%; normalization standard), [M + 2H]2+/b1* (maximum yield, 89%), [M + 2H]2+/y9 (maximum yield, 67%) and [M + 2H]2+/y7 (maximum yield, 5%).
Figure 3
Figure 3. Correlation of laboratory CEmax with the Hammett constant. Substituent a, 4-dimethylamino; b, 4-diethylamino; c, 4-methoxy; d, 4-ethoxy; e, 3,4-dimethoxy; f, 3,5-dimethyl; g, 3-methyl; h, unsubstituted; i, 3,4,5-trimethoxy; j, 3-methoxy; k, 3-methylthio; l, 4-iodo; m, 3-iodo; n, 3-bromo; o, 3-chloro; p, 3-methoxycarbamoyl; q, 4-methoxycarbonyl; r, 3-cyano; s, 4-cyano; t, 3-nitro; and u, 4-nitro. The circle labels are for para substituents: solid red for strong resonance electron-donating and open blue for resonance electron-withdrawing. The solid squares are for meta substituents.
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- 5Anderson, L. and Hunter, C. L. Mol. Cell. Proteomics 2006, 5, 573– 5885https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjsVCmtb8%253D&md5=421ea35aff5f9be237c641cb7aa678c7Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteinsAnderson, Leigh; Hunter, Christie L.Molecular and Cellular Proteomics (2006), 5 (4), 573-588CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)Quant. LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quant. data, demonstrating with-in-run coeffs. of variation (CVs) (n = 10) of 2-22% (78% of assays had CV <10%). A no. of peptides gave CVs in the range 2-7% in five expts. (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coeffs. (R2) of 0.995 and 0.989, resp. Abs. analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 μg/mL) indicates that proteins down to the μg/mL level can be quantitated in plasma with minimal sample prepn., yielding a dynamic range of 4.5 orders of magnitude in a single expt. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope stds. and capture by anti-peptide antibodies (SISCAPA)).
- 6Wolf-Yadlin, A., Hautaniemi, S., Lauffenburger, D. A. and White, F. M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5860– 5865There is no corresponding record for this reference.
- 7Keshishian, H., Addona, T., Burgess, M., Kuhn, E. and Carr, S. A. Mol. Cell. Proteomics 2007, 6, 2212– 22297https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXksF2hsw%253D%253D&md5=e84a98b27604eef97c9eb2c7a1346f6aQuantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilutionKeshishian, Hasmik; Addona, Terri; Burgess, Michael; Kuhn, Eric; Carr, Steven A.Molecular and Cellular Proteomics (2007), 6 (12), 2212-2229CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quant. assays optimized for the selective detection of target proteins. Many protein biomarkers of clin. currency are present at or below the nanogram/mL range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope diln. mass spectrometry, the authors describe here the development of quant., multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/mL range with percent coeffs. of variation from 3% to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addn. of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quant. multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concns. of spiked protein in plasma, the authors detd. that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/mL range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/mL range. These levels of assay performance represent up to a 1000-fold improvement compared with direct anal. of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatog. at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quant. MS-based assays of low level proteins in blood.
- 8Lange, V., Malmstrom, J. A., Didion, J., King, N. L., Johansson, B. P., Schafer, J., Rameseder, J., Wong, C. H., Deutsch, E. W., Brusniak, M. Y., Buhlmann, P., Bjorck, L., Domon, B. and Aebersold, R. Mol. Cell. Proteomics 2008, 7, 1489– 15008https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtVSrs7vO&md5=23244501fc3a8219b4b4ed69b44c8210Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoringLange, Vinzenz; Malmstrom, Johan A.; Didion, John; King, Nichole L.; Johansson, Bjorn P.; Schafer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W.; Brusniak, Mi-Youn; Buhlmann, Peter; Bjorck, Lars; Domon, Bruno; Aebersold, RuediMolecular and Cellular Proteomics (2008), 7 (8), 1489-1500CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)In many studies, particularly in the field of systems biol., it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such expts. has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quant. approach by which predetd. protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quant. Anal. by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amts. of plasma. The resulting quant. protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.
- 9Wysocki, V. H., Tsaprailis, G., Smith, L. L. and Breci, L. A. J. Mass Spectrom. 2000, 35, 1399– 14069https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXltFWrtQ%253D%253D&md5=9fc73aee0031d7a277a4f3a80ca00cf4Mobile and localized protons: a framework for understanding peptide dissociationWysocki, Vicki H.; Tsaprailis, George; Smith, Lori L.; Breci, Linda A.Journal of Mass Spectrometry (2000), 35 (12), 1399-1406CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)Protein identification and peptide sequencing by tandem mass spectrometry requires knowledge of how peptides fragment in the gas phase, specifically which bonds are broken and where the charge(s) resides in the products. For many peptides, cleavage at the amide bonds dominate, producing a series of ions that are designated b and y. For other peptides, enhanced cleavage occurs at just one or two amino acid residues. Surface-induced dissocn., along with gas-phase collision-induced dissocn. performed under a variety of conditions, has been used to refine the general "mobile proton" model and to det. how and why enhanced cleavages occur at aspartic acid residues and protonated histidine residues. Enhanced cleavage at acidic residues occurs when the charge is unavailable to the peptide backbone or the acidic side-chain. The acidic H of the side-chain then serves to initiate cleavage at the amide bond immediately C-terminal to Asp (or Glu), producing an anhydride. In contrast, enhanced cleavage occurs at His when the His side-chain is protonated, turning His into a weak acid that can initiate backbone cleavage by transferring a proton to the backbone. This allows the nucleophilic nitrogen of the His side-chain to attack and form a cyclic structure that is different from the "typical" backbone cleavage structures.
- 10Bleiholder, C., Osburn, S., Williams, T. D., Suhai, S., Van Stipdonk, M., Harrison, A. G. and Paizs, B. J. Am. Chem. Soc. 2008, 130, 17774– 17789There is no corresponding record for this reference.
- 11Wells, J. M. and McLuckey, S. A. Methods Enzymol. 2005, 402, 148– 18511https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XosFeruro%253D&md5=849f2e36d0a18d1ed3c716025970cba6Collision-induced dissociation (CID) of peptides and proteinsWells, J. Mitchell; McLuckey, Scott A.Methods in Enzymology (2005), 402 (Biological Mass Spectrometry), 148-185CODEN: MENZAU; ISSN:0076-6879. (Elsevier)A review. The most commonly used activation method in the tandem mass spectrometry (MS) of peptides and proteins is energetic collisions with a neutral target gas. The overall process of collisional activation followed by fragmentation of the ion is commonly referred to as collision-induced dissocn. (CID). The structural information that results from CID of a peptide or protein ion is highly dependent on the conditions used to effect CID. These include, for example, the relative translational energy of the ion and target, the nature of the target, the no. of collisions that is likely to take place, and the observation window of the app. This chapter summarizes the key exptl. parameters in the CID of peptide and protein ions, as well as the conditions that tend to prevail in the most commonly employed tandem mass spectrometers.
- 12Huang, Y., Triscari, J. M., Tseng, G. C., Pasa-Tolic, L., Lipton, M. S., Smith, R. D. and Wysocki, V. H. Anal. Chem. 2005, 77, 5800– 581312https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXotVSlsbk%253D&md5=b7a412931e70727b82c21993a5c58423Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation PatternsHuang, Yingying; Triscari, Joseph M.; Tseng, George C.; Pasa-Tolic, Ljiljana; Lipton, Mary S.; Smith, Richard D.; Wysocki, Vicki H.Analytical Chemistry (2005), 77 (18), 5800-5813CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Data mining was performed on 28,330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chem. interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissocn. patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliph. residues (Ile, Val, Leu) are obsd. in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are obsd. in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns obsd. expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities.
- 13Huang, Y., Tseng, G. C., Yuan, S., Pasa-Tolic, L., Lipton, M. S., Smith, R. D. and Wysocki, V. H. J. Proteome Res. 2008, 7, 70– 7913https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlygsr%252FK&md5=e4c2967f5d64fdeea40fa9c9a6db85d2A Data-Mining Scheme for Identifying Peptide Structural Motifs Responsible for Different MS/MS Fragmentation Intensity PatternsHuang, Yingying; Tseng, George C.; Yuan, Shinsheng; Pasa-Tolic, Ljiljana; Lipton, Mary S.; Smith, Richard D.; Wysocki, Vicki H.Journal of Proteome Research (2008), 7 (1), 70-79CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Although tandem mass spectrometry (MS/MS) has become an integral part of proteomics, intensity patterns in MS/MS spectra are rarely weighted heavily in most widely used algorithms because they are not yet fully understood. Here a knowledge mining approach is demonstrated to discover fragmentation intensity patterns and elucidate the chem. factors behind such patterns. Fragmentation intensity information from 28 330 ion trap peptide MS/MS spectra of different charge states and sequences went through unsupervised clustering using a penalized K-means algorithm. Without any prior chem. assumptions, four clusters with distinctive fragmentation patterns were obtained. A decision tree was generated to investigate peptide sequence motif and charge state status that caused these fragmentation patterns. This data-mining scheme is generally applicable for any large data sets. It bypasses the common prior knowledge constraints and reports on the overall peptide fragmentation behavior. It improves the understanding of gas-phase peptide dissocn. and provides a foundation for new or improved protein identification algorithms.
- 14Shi, Y., Bajrami, B. and Yao, X. Anal. Chem. 2009, 81, 6438– 6448There is no corresponding record for this reference.
- 15Regnier, F. E. and Julka, S. Proteomics 2006, 6, 3968– 3979There is no corresponding record for this reference.
- 16Summerfield, S. G., Bolgar, M. S. and Gaskell, S. J. J. Mass Spectrom. 1997, 32, 225– 231There is no corresponding record for this reference.
- 17Summerfield, S. G., Steen, H., O’Malley, M. and Gaskell, S. J. Int. J. Mass Spectrom. 1999, 188, 95– 103There is no corresponding record for this reference.
- 18Yalcin, T., Gabryelski, W. and Li, L. J. Mass Spectrom. 1998, 33, 543– 553There is no corresponding record for this reference.
- 19Van Der Rest, G., He, F., Emmett, M. R., Marshall, A. G. and Gaskell, S. J. J. Am. Soc. Mass Spectrom. 2001, 12, 288– 295There is no corresponding record for this reference.
- 20Wang, D., Kalume, D., Pickart, C., Pandey, A. and Cotter, R. J. Anal. Chem. 2006, 78, 3681– 3687There is no corresponding record for this reference.
- 21Wang, D., Fang, S. and Wohlhueter, R. M. Anal. Chem. 2009, 81, 1893– 1900There is no corresponding record for this reference.
- 22Yao, X., Diego, P., Ramos, A. A. and Shi, Y. Anal. Chem. 2008, 80, 7383– 7391There is no corresponding record for this reference.
- 23Savitski, M. M., Kjeldsen, F., Nielsen, M. L., Garbuzynskiy, S. O., Galzitskaya, O. V., Surin, A. K. and Zubarev, R. A. Angew. Chem., Int. Ed. 2007, 46, 1481– 1484There is no corresponding record for this reference.
- 24Abboud, J. L. M., Mo, O., De Paz, J. L. G., Yanez, M., Esseffar, M., Bouab, W., El-Mouhtadi, M., Mokhlisse, R. and Ballesteros, E. J. Am. Chem. Soc. 1993, 115, 12468– 12476There is no corresponding record for this reference.
- 25Tsaprailis, G., Nair, H., Zhong, W., Kuppannan, K., Futrell, J. H. and Wysocki, V. H. Anal. Chem. 2004, 76, 2083– 2094There is no corresponding record for this reference.
- 26Hansch, C., Leo, A. and Taft, R. W. Chem. Rev. 1991, 91, 165– 19526https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXhs1ehsLo%253D&md5=9fc814cd57c47680a5213f3438037800A survey of Hammett substituent constants and resonance and field parametersHansch, Corwin; Leo, A.; Taft, R. W.Chemical Reviews (Washington, DC, United States) (1991), 91 (2), 165-95CODEN: CHREAY; ISSN:0009-2665.Included in this review is an anal. of newer methods which can supplant this classic procedure for detn. of the title consts., 283 refs.
- 27Pfeifer, T., Schierhorn, A., Friedemann, R., Jakob, M., Frank, R., Schutkowski, M. and Fischer, G. J. Mass Spectrom. 1997, 32, 1064– 1071There is no corresponding record for this reference.
- 28Mirzaei, H. and Regnier, F. Anal. Chem. 2006, 78, 4175– 4183There is no corresponding record for this reference.
- 29Williams, D. K., Jr., Meadows, C. W., Bori, I. D., Hawkridge, A. M., Comins, D. L. and Muddiman, D. C. J. Am. Chem. Soc. 2008, 130, 2122– 2123There is no corresponding record for this reference.
- 30Hsu, J.-L., Huang, S.-Y., Shiea, J.-T., Huang, W.-Y. and Chen, S.-H. J. Proteome Res. 2005, 4, 101– 10830https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtFWqsr%252FK&md5=24d15f458030df9cb2df9df759ba9e95Beyond Quantitative Proteomics: Signal Enhancement of the a1 Ion as a Mass Tag for Peptide Sequencing Using Dimethyl LabelingHsu, Jue-Liang; Huang, Sheng-Yu; Shiea, Jen-Taie; Huang, Wen-Ying; Chen, Shu-HuiJournal of Proteome Research (2005), 4 (1), 101-108CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Stable isotope-based di-Me labeling that produces a di-Me labeled terminal amine or a monomethylated proline N-terminus by reductive methylation (Anal. Chem. 2003, 75, 6843-6852) was reported as a promising strategy for global quant. proteomics because of the simplicity of the process and its fast and complete reaction. This labeling strategy provides a signal enhancement for the produced a1 ions, which are usually hard to detect among most of the nonderivatized fragments. To assist peptide sequencing, in this study, the enhanced a1 ion produced under either collision induced dissocn. (CID) or post source decay (PSD) modes was further characterized and applied as a mass tag for fingerprinting the identity of N-terminal amino acid. On the basis of the anal. of std. peptides, tryptic digests of Hb and cell lysates, it was proved that such signal enhancement occurred to a1 ions derived from all 20 of the amino acids residues and this phenomenon was explained based the formation of stable quaternary immonium ions. Accurate detn. of a1 ions was shown to increase the chance for peptide de novo sequencing and also provided higher confidence in the scores obtained when identifying a protein through database searching. In addn., the a1 ion was further demonstrated to be used as a universal tag for precursor ion scan in a Q-TOF instrument, leading to a greater no. of peptide ions sequenced. Combined with the capability for differential quantitation, the stable isotope-based di-Me labeling increases the usefulness of the labeling method for MS-based proteomics.
- 31Fu, Q. and Li, L. Anal. Chem. 2005, 77, 7783– 779531https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFOis7fL&md5=599df751d27b143c9eef5ba30d9cae14De Novo Sequencing of Neuropeptides Using Reductive Isotopic Methylation and Investigation of ESI QTOF MS/MS Fragmentation Pattern of Neuropeptides with N-Terminal DimethylationFu, Qiang; Li, LingjunAnalytical Chemistry (2005), 77 (23), 7783-7795CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)A stable-isotope di-Me labeling strategy was previously shown to be a useful tool for quant. proteomics. More recently, N-terminal di-Me labeling was also reported for peptide sequencing in combination with database searching. Here, the authors extend these previous studies by incorporating N-terminal isotopic dimethylation for de novo sequencing of neuropeptides directly from tissue ext. without any genomic information. The authors demonstrated several new sequencing applications of this method in addn. to the identification of the N-terminal residue using the enhanced a1 ion. The isotopic labeling also provides easier and more confident de novo sequencing of peptides by comparing similar MS/MS fragmentation patterns of the isotopically labeled peptide pairs. The current study on neuropeptides shows several distinct fragmentation patterns after N-terminal dimethylation which have not been reported previously. The y(n-1) ion is enhanced in multiply charged peptides and is weak or missing in singly charged peptides. The MS/MS spectra of singly charged peptides are simplified due to the enhanced N-terminal fragments and suppressed internal fragments. The neutral loss of dimethylamine is also obsd. The mechanisms for the above fragmentations are proposed. Finally, the structures of the immonium ion and related ions of Nα, Nε-tetramethylated lysine and Nε-dimethylated lysine are explored.
- 32Locke, S. J., Leslie, A. D., Melanson, J. E. and Pinto, D. M. Rapid Commun. Mass Spectrom. 2006, 20, 1525– 153032https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVartb8%253D&md5=0e6a5927fa3fefbfcea78e1eca23b508Deviation from the mobile proton model in amino-modified peptides: implications for multiple reaction monitoring analysis of peptidesLocke, Steven J.; Leslie, Andrew D.; Melanson, Jeremy E.; Pinto, Devanand M.Rapid Communications in Mass Spectrometry (2006), 20 (10), 1525-1530CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)The study of peptide fragmentation is important to the understanding of chem. processes occurring in the gas phase and the more practical concern of peptide identification for proteomic anal. Using the mobile proton model as a framework, the authors explored the effect of amino-group modifications on peptide fragmentation. Three aldehydes were used to transform the primary amino groups on peptides into either a dimethylamino or a heterocyclic structure (five- or six-membered). The obsd. fragmentation patterns deviated strongly from those obsd. for the analogous underivatized peptides. In particular, the a1 ion was the base peak in most tandem mass spectra of the derivatized peptides. It was obsd. that the a1 ion intensity depends strongly on the N-terminal amino acid, with tyrosine and phenylalanine having the strongest enhancement. Despite the change in fragmentation patterns of the derivatized peptides, they still provided high-quality tandem mass spectra that, in many cases, are more amenable to database searching than the spectra of underivatized peptides. In addn., the reliable presence of the a1 ion facilitated rapid quant. measurements using the multiple reaction monitoring approach.
- 33Desouza, L. V., Taylor, A. M., Li, W., Minkoff, M. S., Romaschin, A. D., Colgan, T. J. and Siu, K. W. J. Proteome Res. 2008, 7, 3525– 353433https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXosFKmsbg%253D&md5=8f1b2405f6537fe6f41269fb28c560b2Multiple Reaction Monitoring of mTRAQ-Labeled Peptides Enables Absolute Quantification of Endogenous Levels of a Potential Cancer Marker in Cancerous and Normal Endometrial TissuesDe Souza, Leroi V.; Taylor, Adrian M.; Li, Wei; Minkoff, Marjorie S.; Romaschin, Alexander D.; Colgan, Terence J.; Siu, K. W. MichaelJournal of Proteome Research (2008), 7 (8), 3525-3534CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)While iTRAQ analyses have proved invaluable for the discovery of potential cancer markers, two outstanding issues that remained were its ineffectiveness to consistently detect specific proteins of interest in a complex sample and to det. the abs. abundance of those proteins. These have been addressed by availability of the mTRAQ reagents (Applied Biosystems, Inc., Foster City, CA) a nonisobaric variant of iTRAQ. We have applied this newly emerging technique to quantify one of our potential markers for endometrial cancer, viz. pyruvate kinase M1/M2. The mTRAQ methodol. relies on multiple reaction monitoring (MRM) to target tryptic peptides from the protein of interest, thus, ensuring maximal opportunity for detection, while the nonisobaric tags enable specific quantification of each version of the labeled peptides through unique MRM transitions conferred by the labels. Known amts. of synthetic peptides tagged with one of the two available mTRAQ labels, when used as quantification stds. in a mixt. with the oppositely labeled tryptically digested sample, permit detn. of the abs. amts. of the corresponding protein in the sample. The ability to label the sample and ref. peptides with either one of the two possible combinations is an inherent advantage of this method, as it provides a means for verification of the reported ratios. In this study, we detd. that the amt. of pyruvate kinase present in the homogenate from a biopsied EmCa tissue sample was 85 nmol/g of total proteins, while the equiv. concn. in the nonmalignant controls was 21-26 nmol/g of total proteins. This approx. 4-fold higher amt. of pyruvate kinase in the cancer sample was further confirmed not only by a direct comparison between the cancer sample and one of the nonmalignant controls, but also independently by an enzyme-linked immunosorbant assay (ELISA). Addnl., the 4-fold higher level of pyruvate kinase amt. in the cancer homogenate reported in this study is considerably higher than the 2-fold higher ratio reported across 20 cancer samples in the discovery phase with the iTRAQ technique, suggesting that there exists a possibility that the dynamic range of ratios detd. by the iTRAQ technique may have been compressed.
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