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Averagine-Scaling Analysis and Fragment Ion Mass Defect Labeling in Peptide Mass Spectrometry Xudong Yao,* Pamela Diego, Alexis A. Ramos, and Yu Shi Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269 A method termed as the averagine-scaling analysis (ASA) is proposed for predictive design and selection of chemical reagents for modifying peptides, as well as for facile mass spectral analysis of peptide fragment ions with increased mass defects. The ASA method scales mass spectral data using the mass of the hypothetical averagine residue as reference. The scaling analysis is used in conjunction with a strategy of fragment ion mass defect labeling (FIMDL) for effectively using the broad, unoccupied mass zones in the low m/z region of mass spectra. The FIMDL approach involves the solution modification of peptide termini with chemical reagents of large mass defects and the gas-phase generation of peptide terminal fragment ions that carry the FIMDL groups. The scaling analysis reveals that iodine has the highest FIMDL efficiency among halogens. Iodine-containing reagents, 4-iodophenylisocyanate and 4-iodophenylisothiocyanate, are used to label primary amines on peptides to demonstrate the scaling analysis. The ASA method successfully distinguishes peptide fragment ions with and without an FIMDL group and specifically and efficiently reduces the data complexity of peptide tandem mass spectra. The combination of ASA with FIMDL extends the instrument suitability for the mass defect analysis from mass spectrometers of ultrahigh mass resolution and accuracy to those of medium ones. This combination is expected to have a profound impact on peptide tandem mass spectrometry. The chemical derivatization of peptides provides a means to introduce quantitation, signal-enhancing or signal-simplifying labels for mass spectrometric analysis.1,2 The N-terminus of peptides presents a readily accessible site for various methods of chemical derivatization such as acylation and reductive alkylation.3 All of the N-terminal modifications result in structural changes of the N-terminal fragment ions, if the modifying group is relatively stable to gas-phase collisions; the modified fragments can be used as marker ions to facilitate the identification and quantitation of peptides and proteins by mass spectrometry. For example, the reductive methylation of N-terminal amines by aldehydes enhances the production of the a1-type ions of peptides by promoting the generation of the bismethylated immonium ions of the first amino acid residues on peptides. Both thiocarbamoylation and * Corresponding author. E-mail:
[email protected]. (1) Leitner, A.; Lindner, W. Proteomics 2006, 6, 5418. (2) Hung, C.-W.; Schlosser, A.; Wei, J.; Lehmann, W. D. Anal. Bioanal. Chem. 2007, 389, 1003. (3) Regnier, F. E.; Julka, S. Proteomics 2006, 6, 3968. 10.1021/ac801096e CCC: $40.75 2008 American Chemical Society Published on Web 09/09/2008
carbamoylation of peptide N-terminal amines also produce modified N-terminal fragment ions.4-7 Modified peptide fragment ions have found various applications in proteome analysis assisting quantitation and identification of proteins and peptides.7-11 However, the specificity of the modified N-terminal ions in the low m/z region is intrinsically limited by the isobaric interfering ions, which are primary and secondary fragments of peptides. These are generated by gas-phase fragmentation of peptide ions upon collisional activation in mass spectrometers and occur for almost every mass unit. One approach for improving the specificity of small fragment ions is to identify “quiet or quasi-quiet” regions that have smaller probabilities of observing fragment ions; this approach has helped the successful development of the tandem mass tag technology.12-15 Another strategy effectively uses the unoccupied mass spaces in a spectrum. It takes cues from the mass property of small fragment ions from phosphorylated peptides. Phosphotyrosine (pY) peptides produce the unique pY immonium ion at m/z 216.043, which can be basically separated from its isobaric interfering ions on mass spectrometers with medium mass resolution and accuracy.16-18 This separation is due to the increased mass defect caused by the attachment of the phosphate group. The mass of every peptide differs characteristically from a round number by a value called the mass defect. The mass defect of the pY immonium ion is significantly larger than those of other isobaric ions, and thus this ion can be used as a marker for (4) Summerfield, S. G.; Bolgar, M. S.; Gaskell, S. J. J. Mass Spectrom. 1997, 32, 225. (5) Yalcin, T.; Gabryelski, W.; Li, L. J. Mass Spectrom. 1998, 33, 543. (6) van der Rest, G.; He, F.; Emmett, M. R.; Marshall, A. G.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 2001, 12, 288. (7) Mason, D. E.; Liebler, D. C. J. Proteome Res. 2003, 2, 265. (8) Hsu, J.-L.; Huang, S.-Y.; Chow, N.-H.; Chen, S.-H. Anal. Chem. 2003, 75, 6843. (9) Fu, Q.; Li, L. Anal. Chem. 2005, 77, 7783. (10) Hsu, J.-L.; Huang, S.-Y.; Shiea, J.-T.; Huang, W.-Y.; Chen, S.-H. J. Proteome Res. 2005, 4, 101. (11) Ji, C.; Guo, N.; Li, L. J. Proteome Res. 2005, 4, 2099. (12) Zhang, Y.; Wolf-Yadlin, A.; Ross, P. L.; Pappin, D. J.; Rush, J.; Lauffenburger, D. A.; White, F. M. Mol. Cell. Proteomics 2005, 4, 1240. (13) Sachon, E.; Mohammed, S.; Bache, N.; Jensen, O. N. Rapid Commun. Mass Spectrom. 2006, 20, 1127. (14) Thompson, A.; Schafer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Neumann, T.; Johnstone, R.; Mohammed, A. K.; Hamon, C. Anal. Chem. 2003, 75, 1895. (15) Dayon, L.; Hainard, A.; Licker, V.; Turck, N.; Kuhn, K.; Hochstrasser, D. F.; Burkhard, P. R.; Sanchez, J. C. Anal. Chem. 2008, 80, 2921. (16) Steen, H.; Kuester, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem. 2001, 73, 1440. (17) Salek, M.; Alonso, A.; Pipkorn, R.; Lehmann, W. D. Anal. Chem. 2003, 75, 2724. (18) Shi, Y.; Yao, X. Anal. Chem. 2007, 79, 8454.
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selective detection of pY peptides in mixtures at the mass resolution and accuracy of contemporary time-of-flight (TOF) mass spectrometers. In another instance, peptides containing iodinylated tyrosine residues are specifically identified based on the iodinecontaining tyrosine immonium ions that are well separated from the isobaric interfering ions.19 Together with bromotryptophancontaining polypeptides,20,21 they are naturally occurring examples for the principle of the mass defect labeling (MDL).22-24 The biosynthesis of proteins uses only a restricted number of amino acids, as well as a preferred range of sequences. Therefore, a limited number of elements control the population of the mass defect of proteins and peptides on the mass scale, providing opportunities in monoisotopic mass determination by mass spectrometry,25,26 e.g., filtering nonpeptide signals in peptide mass spectrometry27 and selectively analyzing peptides that carry posttranslational modifications.28,29 These methods take the advantage of the fact that mass defects of peptides cluster discretely, resulting in zones in a spectrum that are not occupied by any native peptide ions, the so-called forbidden zone.27,30,31 These zones can host peptide fragment ions with the increased mass defects of minimal interference. MDL also uses this fact for tagging peptides and proteins with reagents that contain atoms of large mass defects.22-24 MDL reagents often contain atoms with the atomic mass number between 17 and 77, such as Cl and Br;23 for example, 2,4-dibromo(2′-iodo)acetanilide has been used to modify peptide cysteine and the modified peptides are subjected to matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry.22 The changes in the mass defect of cysteine-containing peptides allow specific identification of these peptides and increase the number of identified proteins.22 Acylation and reductive alkylation reactions have also been proposed to tag primary amines on polypeptides with MDL reagents, such as the succinimidyl ester of 5-bromo-3-pyridylacetic acid and 4-bromobenzaldehyde, in a method that uses isotope-differentiated binding energy shift tags.23,24 However, the stability of the later tagging groups on peptides upon collisional activation has yet been thoroughly investigated. Because MDL results in only a small increase in the mass defect of peptides, the differentiation of mass spectrometric peaks for the tagged peptide ions from the isobaric native ones, based (19) Salek, M.; Lehmann, W. D. Proteomics 2005, 5, 351. (20) Steen, H.; Mann, M. Anal. Chem. 2002, 74, 6230. (21) Nair, S. S.; Nilsson, C. L.; Emmett, M. R.; Schaub, T. M.; Gowd, K. H.; Thakur, S. S.; Krishnan, K. S.; Balaram, P.; Marshall, A. G. Anal. Chem. 2006, 78, 8082. (22) Hernandez, H.; Niehauser, S.; Boltz, S. A.; Gawandi, V.; Phillips, R. S.; Amster, I. J. Anal. Chem. 2006, 78, 3417. (23) Hall, M. P.; Ashrafi, S.; Obegi, I.; Petesch, R.; Peterson, J. N.; Schneider, L. V. J. Mass Spectrom. 2003, 38, 809. (24) Hall, M. P.; Schneider, L. V. Expert Rev. Proteomics 2004, 1, 421. (25) Yergey, J.; Heller, D.; Hansen, G.; Cotter, R. J.; Fenselau, C. Anal. Chem. 1983, 55, 353. (26) Liu, T.; Belov, M. E.; Jaitly, N.; Qian, W.-J.; Smith, R. D. Chem. Rev. 2007, 107, 3621. (27) Dodds, E. D.; An, H. J.; Hagerman, P. J.; Lebrilla, C. B. J. Proteome Res. 2006, 5, 1195. (28) Lehmann, W. D.; Bohne, A.; Von der Lieth, C.-W. J. Mass Spectrom. 2000, 35, 1335. (29) Bruce, C.; Shifman, M. A.; Miller, P.; Gulcicek, E. E. Anal. Chem. 2006, 78, 4374. (30) Mann, M. In Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, May 21-26, 1995, p 639. (31) Frahm, J. L.; Howard, B. E.; Heber, S.; Muddiman, D. C. J. Mass Spectrom. 2006, 41, 281.
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on their mass defects, requires mass spectrometers with ultrahigh mass accuracy and resolution; FT mass spectrometers like magnetic ion cyclotron resonance (ICR) or electrostatic Orbitrap are thus the instruments of choice. Relative changes in the mass defect, however, are more significant to small ions. Therefore, medium mass resolution and mass accuracy, which can be achieved on TOF mass spectrometers and some ion trap and quadrupole instruments operated under certain modes, are sufficient to distinguish minute differences in mass defects of small ions, e.g.,
