Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

  • Brian L. Frey
  • Daniel T. Ladror
  • Samuel B. Sondalle
  • Casey J. Krusemark
  • April L. Jue
  • Joshua J. Coon
  • Lloyd M. SmithEmail author
Focus: Electron Transfer Dissociation: Research Article


The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z  >  2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

Key words

Peptide derivatization Peptide carboxylic acids Carboxyl group derivatization Fixed charge modification Tertiary amine Charge state Peptide fragmentation Electron transfer dissociation Amino acid sequence Sequence coverage Ribosomal protein Proteomics Mass spectrometry 



The authors thank Amelia (Mia) Zutz for performing labeling reactions on the neurotensin peptide standards, M. Violet Lee for compiling the precursor charge state data, and A. J. Bureta for help with figure illustrations. The authors are grateful to Professor Toshifumi Inada at Nagoya University, Nagoya, Japan for the gift of the YIT613 FLAG-tagged yeast strain. This work was supported by the National Institutes of Health: NIGMS Program Project P01GM081629, R01 GM080148, and NHGRI Center of Excellence in Genomic Science 1P50HG004952.


  1. 1.
    Smith, L.M., Kelleher, N.L.: Proteoform: a single term describing protein complexity. Nat. Methods 10, 186–187 (2013)CrossRefGoogle Scholar
  2. 2.
    Krusemark, C.J., Frey, B.L., Smith, L.M., Belshaw, P.J.: Complete chemical modification of amine and acid functional groups of peptides and small proteins. Methods Mol. Biol. 753, 77–91 (2011)CrossRefGoogle Scholar
  3. 3.
    Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 101, 9528–9533 (2004)CrossRefGoogle Scholar
  4. 4.
    Pitteri, S.J., Chrisman, P.A., Hogan, J.M., McLuckey, S.A.: Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*. Anal. Chem. 77, 1831–1839 (2005)CrossRefGoogle Scholar
  5. 5.
    Good, D.M., Wirtala, M., McAlister, G.C., Coon, J.J.: Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 6, 1942–1951 (2007)CrossRefGoogle Scholar
  6. 6.
    Wiesner, J., Premsler, T., Sickmann, A.: Application of electron transfer dissociation (ETD) for the analysis of post-translational modifications. Proteomics 8, 4466–4483 (2008)CrossRefGoogle Scholar
  7. 7.
    Swaney, D.L., McAlister, G.C., Coon, J.J.: Decision tree-driven tandem mass spectrometry for shotgun proteomics. Nat. Methods 5, 959–964 (2008)CrossRefGoogle Scholar
  8. 8.
    Xia, Y., Gunawardena, H.P., Erickson, D.E., McLuckey, S.A.: Effects of cation charge-site identity and position on electron-transfer dissociation of polypeptide cations. J. Am. Chem. Soc. 129, 12232–12243 (2007)CrossRefGoogle Scholar
  9. 9.
    Xia, Y., Han, H., McLuckey, S.A.: Activation of intact electron-transfer products of polypeptides and proteins in cation transmission mode ion/ion reactions. Anal. Chem. 80, 1111–1117 (2008)CrossRefGoogle Scholar
  10. 10.
    Madsen, J.A., Brodbelt, J.S.: Simplifying fragmentation patterns of multiply charged peptides by n-terminal derivatization and electron transfer collision activated dissociation. Anal. Chem. 81, 3645–3653 (2009)CrossRefGoogle Scholar
  11. 11.
    Ledvina, A.R., Beauchene, N.A., McAlister, G.C., Syka, J.E.P., Schwartz, J.C., Griep-Raming, J., Westphall, M.S., Coon, J.J.: Activated-ion electron transfer dissociation improves the ability of electron transfer dissociation to identify peptides in a complex mixture. Anal. Chem. 82, 10068–10074 (2010)CrossRefGoogle Scholar
  12. 12.
    Iavarone, A.T., Jurchen, J.C., Williams, E.R.: Supercharged protein and peptide ions formed by electrospray ionization. Anal. Chem. 73, 1455–1460 (2001)CrossRefGoogle Scholar
  13. 13.
    Lomeli, S.H., Yin, S., Loo, R.R.O., Loo, J.A.: Increasing charge while preserving noncovalent protein complexes for ESI-MS. J. Am. Soc. Mass Spectrom. 20, 593–596 (2009)CrossRefGoogle Scholar
  14. 14.
    Kjeldsen, F., Giessing, A.M.B., Ingrell, C.R., Jensen, O.N.: Peptide sequencing and characterization of post-translational modifications by enhanced ion-charging and liquid chromatography electron-transfer dissociation tandem mass spectrometry. Anal. Chem. 79, 9243–9252 (2007)CrossRefGoogle Scholar
  15. 15.
    Thompson, A., Schafer, J., Kuhn, K., Kienle, S., Schwarz, J., Schmidt, G., Neumann, T., Johnstone, R., Mohammed, A.K., Hamon, C.: Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003)CrossRefGoogle Scholar
  16. 16.
    Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A., Pappin, D.J.: Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteom. 3, 1154–1169 (2004)CrossRefGoogle Scholar
  17. 17.
    Mirzaei, H., Regnier, F.: Enhancing electrospray ionization efficiency of peptides by derivatization. Anal. Chem. 78, 4175–4183 (2006)CrossRefGoogle Scholar
  18. 18.
    Chamot-Rooke, J., van der Rest, G., Dalleu, A., Bay, S., Lemoine, J.: The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides. J. Am. Soc. Mass Spectrom. 18, 1405–1413 (2007)CrossRefGoogle Scholar
  19. 19.
    Xiang, F., Ye, H., Chen, R.B., Fu, Q., Li, L.J.: N, N-dimethyl leucines as novel isobaric tandem mass tags for quantitative proteomics and peptidomics. Anal. Chem. 82, 2817–2825 (2010)CrossRefGoogle Scholar
  20. 20.
    Lu, Y.L., Zhou, X., Stemmer, P.M., Reid, G.E.: Sulfonium ion derivatization, isobaric stable isotope labeling and data dependent CID- and ETD-MS/MS for enhanced phosphopeptide quantitation, identification and phosphorylation site characterization. J. Am. Soc. Mass Spectrom. 23, 577–593 (2012)CrossRefGoogle Scholar
  21. 21.
    Wuhr, M., Haas, W., McAlister, G.C., Peshkin, L., Rad, R., Kirschner, M.W., Gygi, S.P.: Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster. Anal. Chem. 84, 9214–9221 (2012)Google Scholar
  22. 22.
    Hennrich, M.L., Boersema, P.J., van den Toorn, H., Mischerikow, N., Heck, A.J., Mohammed, S.: Effect of chemical modifications on peptide fragmentation behavior upon electron transfer induced dissociation. Anal. Chem. 81, 7814–7822 (2009)CrossRefGoogle Scholar
  23. 23.
    Hsu, J.L., Huang, S.Y., Chow, N.H., Chen, S.H.: Stable-isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75, 6843–6852 (2003)CrossRefGoogle Scholar
  24. 24.
    Hsu, J.-L., Huang, S.-Y., Shiea, J.-T., Huang, W.-Y., Chen, S.-H.: Beyond quantitative proteomics: signal enhancement of the a1 ion as a mass tag for peptide sequencing using dimethyl labeling. J. Proteome Res. 4, 101–108 (2005)CrossRefGoogle Scholar
  25. 25.
    Fu, Q., Li, L.: De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation. Anal. Chem. 77, 7783–7795 (2005)CrossRefGoogle Scholar
  26. 26.
    Melanson, J.E., Avery, S.L., Pinto, D.M.: High-coverage quantitative proteomics using amine-specific isotopic labeling. Proteomics 6, 4466–4474 (2006)CrossRefGoogle Scholar
  27. 27.
    Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A.J.R.: Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009)CrossRefGoogle Scholar
  28. 28.
    Krusemark, C.J., Ferguson, J.T., Wenger, C.D., Kelleher, N.L., Belshaw, P.J.: Global amine and acid functional group modification of proteins. Anal. Chem. 80, 713–720 (2008)CrossRefGoogle Scholar
  29. 29.
    Kulevich, S.E., Frey, B.L., Kreitinger, G., Smith, L.M.: Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis. Anal. Chem. 82, 10135–10142 (2010)CrossRefGoogle Scholar
  30. 30.
    Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., Aebersold, R.: Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999)CrossRefGoogle Scholar
  31. 31.
    Ren, D.Y., Julka, S., Inerowicz, H.D., Regnier, F.E.: Enrichment of cysteine-containing peptides from tryptic digests using a quaternary amine tag. Anal. Chem. 76, 4522–4530 (2004)CrossRefGoogle Scholar
  32. 32.
    Yi, E.C., Li, X.J., Cooke, K., Lee, H., Raught, B., Page, A., Aneliunas, V., Hieter, P., Goodlett, D.R., Aebersold, R.: Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme. Proteomics 5, 380–387 (2005)CrossRefGoogle Scholar
  33. 33.
    Williams Jr., D.K., Meadows, C.W., Bori, I.D., Hawkridge, A.M., Comins, D.L., Muddiman, D.C.: Synthesis, characterization, and application of iodoacetamide derivatives utilized for the ALiPHAT strategy. J. Am. Chem. Soc. 130, 2122–2123 (2008)CrossRefGoogle Scholar
  34. 34.
    Wang, J., Zhang, J., Arbogast, B., Maier, C.S.: Tandem mass spectrometric characterization of thiol peptides modified by the chemoselective cationic sulfhydryl reagent (4-iodobutyl)triphenylphosphonium. J. Am. Soc. Mass Spectrom. 22, 1771–1783 (2011)CrossRefGoogle Scholar
  35. 35.
    Ueberheide, B.M., Fenyo, D., Alewood, P.F., Chait, B.T.: Rapid, sensitive analysis of cysteine rich peptide venom components. Proc. Natl. Acad. Sci. U.S.A. 106, 6910–6915 (2009)CrossRefGoogle Scholar
  36. 36.
    Vasicek, L., Brodbelt, J.S.: Enhanced electron transfer dissociation through fixed charge derivatization of cysteines. Anal. Chem. 81, 7876–7884 (2009)CrossRefGoogle Scholar
  37. 37.
    Reid, G.E., Roberts, K.D., Simpson, R.J., O'Hair, R.A.: Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 16, 1131–1150 (2005)CrossRefGoogle Scholar
  38. 38.
    Xu, Y.W., Zhang, L.J., Lu, H.J., Yang, P.Y.: Mass spectrometry analysis of phosphopeptides after peptide carboxy group derivatization. Anal. Chem. 80, 8324–8328 (2008)CrossRefGoogle Scholar
  39. 39.
    Zhang, L.J., Xu, Y.W., Lu, H.J., Yang, P.Y.: Carboxy group derivatization for enhanced electron-transfer dissociation mass spectrometric analysis of phosphopeptides. Proteomics 9, 4093–4097 (2009)CrossRefGoogle Scholar
  40. 40.
    Qiao, X.Q., Sun, L.L., Chen, L.F., Zhou, Y.A., Yang, K.G., Liang, Z., Zhang, L.H., Zhang, Y.K.: Piperazines for peptide carboxyl group derivatization: effect of derivatization reagents and properties of peptides on signal enhancement in matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 25, 639–646 (2011)CrossRefGoogle Scholar
  41. 41.
    Zhang, J.M., Al-Eryani, R., Ball, H.L.: Mass spectrometry analysis of 2-nitrophenylhydrazine carboxy derivatized peptides. J. Am. Soc. Mass Spectrom. 22, 1958–1967 (2011)CrossRefGoogle Scholar
  42. 42.
    Ko, B.J., Brodbelt, J.S.: Enhanced electron transfer dissociation of peptides modified at C-terminus with fixed charges. J. Am. Soc. Mass Spectrom. 23, 1991–2000 (2012)CrossRefGoogle Scholar
  43. 43.
    Frey, B.L., Krusemark, C.J., Ledvina, A.R., Coon, J.J., Belshaw, P.J., Smith, L.M.: Ion–ion reactions with fixed-charge modified proteins to produce ions in a single, very high charge state. Int. J. Mass Spectrom. 276, 136–143 (2008)CrossRefGoogle Scholar
  44. 44.
    Krusemark, C.J., Frey, B.L., Belshaw, P.J., Smith, L.M.: Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization. J. Am. Soc. Mass Spectrom. 20, 1617–1625 (2009)CrossRefGoogle Scholar
  45. 45.
    Inada, T., Winstall, E., Tarun, S.Z., Yates, J.R., Schieltz, D., Sachs, A.B.: One-step affinity purification of the yeast ribosome and its, associated proteins and mRNAs. RNA 8, 948–958 (2002)CrossRefGoogle Scholar
  46. 46.
    Simons, S.P., McLellan, T.J., Aeed, P.A., Zaniewski, R.P., Desbonnet, C.R., Wondrack, L.M., Marr, E.S., Subashi, T.A., Dougherty, T.J., Xu, Z.Y., Wang, I.K., LeMotte, P.K., Maguire, B.A.: Purification of the large ribosomal subunit via its association with the small subunit. Anal. Biochem. 395, 77–85 (2009)CrossRefGoogle Scholar
  47. 47.
    Krokhin, O.V., Spicer, V.: Peptide retention standards and hydrophobicity indexes in reversed-phase high-performance liquid chromatography of peptides. Anal. Chem. 81, 9522–9530 (2009)CrossRefGoogle Scholar
  48. 48.
    Schnier, P.D., Gross, D.S., Williams, E.R.: On the Maximum charge-state and proton-transfer reactivity of peptide and protein ions formed by electrospray-ionization. J. Am. Soc. Mass Spectrom. 6, 1086–1097 (1995)CrossRefGoogle Scholar
  49. 49.
    Coon, J.J., Ueberheide, B., Syka, J.E.P., Dryhurst, D.D., Ausio, J., Shabanowitz, J., Hunt, D.F.: Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 102, 9463–9468 (2005)CrossRefGoogle Scholar
  50. 50.
    Cooper, H.J., Hudgins, R.R., Hakansson, K., Marshall, A.G.: Characterization of amino acid side chain losses in electron capture dissociation. J. Am. Soc. Mass Spectrom. 13, 241–249 (2002)CrossRefGoogle Scholar
  51. 51.
    Rumachik, N.G., McAlister, G.C., Russell, J.D., Bailey, D.J., Wenger, C.D., Coon, J.J.: Characterizing peptide neutral losses induced by negative electron-transfer dissociation (NETD). J. Am. Soc. Mass Spectrom. 23, 718–727 (2012)CrossRefGoogle Scholar
  52. 52.
    Iavarone, A.T., Paech, K., Williams, E.R.: Effects of charge state and cationizing agent on the electron capture dissociation of a peptide. Anal. Chem. 76, 2231–2238 (2004)CrossRefGoogle Scholar
  53. 53.
    Jones, A.W., Cooper, H.J.: Dissociation techniques in mass spectrometry-based proteomics. Analyst 136, 3419–3429 (2011)CrossRefGoogle Scholar
  54. 54.
    Huang, Y.Y., Triscari, J.M., Tseng, G.C., Pasa-Tolic, L., Lipton, M.S., Smith, R.D., Wysocki, V.H.: Statistical characterization of the charge state and residue dependence of low-energy CID peptide dissociation patterns. Anal. Chem. 77, 5800–5813 (2005)CrossRefGoogle Scholar
  55. 55.
    Jones, J.W., Sasaki, T., Goodlett, D.R., Turecek, F.: Electron capture in spin-trap capped peptides. An experimental example of ergodic dissociation in peptide cation-radicals. J. Am. Soc. Mass Spectrom. 18, 432–444 (2007)CrossRefGoogle Scholar
  56. 56.
    Li, X.J., Cournoyer, J.J., Lin, C., O'Connor, P.B.: The effect of fixed charge modifications on electron capture dissociation. J. Am. Soc. Mass Spectrom. 19, 1514–1526 (2008)CrossRefGoogle Scholar
  57. 57.
    Chung, T.W., Turecek, F.: Amplified histidine effect in electron-transfer dissociation of histidine-rich peptides from histatin 5. Int. J. Mass Spectrom. 306, 99–107 (2011)CrossRefGoogle Scholar
  58. 58.
    Chung, T.W., Turecek, F.: Selecting fixed-charge groups for electron-based peptide dissociations—a computational study of pyridinium tags. Int. J. Mass Spectrom. 276, 127–135 (2008)CrossRefGoogle Scholar
  59. 59.
    Chung, T.W., Moss, C.L., Zimnicka, M., Johnson, R.S., Moritz, R.L., Turecek, F.: Electron-capture and -transfer dissociation of peptides tagged with tunable fixed-charge groups: structures and dissociation energetics. J. Am. Soc. Mass Spectrom. 22, 13–30 (2011)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2013

Authors and Affiliations

  • Brian L. Frey
    • 1
  • Daniel T. Ladror
    • 1
  • Samuel B. Sondalle
    • 1
  • Casey J. Krusemark
    • 1
  • April L. Jue
    • 1
  • Joshua J. Coon
    • 1
    • 2
    • 3
  • Lloyd M. Smith
    • 1
    • 3
    Email author
  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of Biomolecular ChemistryUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Genome Center of WisconsinUniversity of Wisconsin-MadisonMadisonUSA

Personalised recommendations