Advertisement

Complete Chemical Modification of Amine and Acid Functional Groups of Peptides and Small Proteins

  • Casey J. KrusemarkEmail author
  • Brian L. Frey
  • Lloyd M. Smith
  • Peter J. Belshaw
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 753)

Abstract

The chemical modification of protein thiols by reduction and alkylation is common in the preparation of proteomic samples for analysis by mass spectrometry (MS). Modification at other functional groups has received less attention in MS-based proteomics. Amine modification (Lys, N-termini) by reductive dimethylation or by acylation (e.g., iTRAQ labeling) has recently gained some popularity in peptide-based approaches (bottom-up MS). Modification at acidic groups (Asp, Glu, C-termini) has been explored very minimally. Here, we describe a sequential labeling strategy that enables complete modification of thiols, amines, and acids on peptides or small intact proteins. This method includes (1) the reduction and alkylation of thiols, (2) the reductive dimethylation of amines, and (3) the amidation of acids with any of several amines. This chemical modification scheme offers several options both for the incorporation of stable isotopes for relative quantification and for improving peptides or proteins as MS analytes.

Key words

Mass spectrometry (MS) stable isotope labeling acylation dimethylation amidation proteomics protein derivatization peptide derivatization 

Notes

Acknowledgments

This work was supported by the National Heart Lung Blood Institute (NHLBI) Proteomics Program N01-HV-28182 and NIH grant P01GM081629. We wish to thank Samuel Sondalle for preparing and analyzing the modified neurotensin samples.

References

  1. 1.
    Krusemark, C. J., Ferguson, J. T., Wenger, C. D., Kelleher, N. L., Belshaw, P. J. (2008) Global amine and acid functional group modification of proteins. Anal Chem 80, 713–720.PubMedCrossRefGoogle Scholar
  2. 2.
    Krusemark, C. J., Frey, B. L., Belshaw, P. J., Smith, L. M. (2009) Modifying the charge state distribution of proteins in electrospray ionization mass spectrometry by chemical derivatization. J Am Soc Mass Spectrom 20, 1617–1625.PubMedCrossRefGoogle Scholar
  3. 3.
    Frey, B. L., Krusemark, C. J., Belshaw, P. J., Smith, L. M. (2008) Ion-ion reactions with fixed-charge modified proteins produce ion in a single, very high charge state. Int J Mass Spectrom 276, 136–143.PubMedCrossRefGoogle Scholar
  4. 4.
    Zhao, J. Y., Waldron, K. C., Miller, J., Zhang, J. Z., Harke, H., Dovicki, N. J. (1992) Attachment of a single fluorescent label to peptides for determination by capillary zone electrophoresis. J Chromatogr 608, 239–242.PubMedCrossRefGoogle Scholar
  5. 5.
    Chait, B. T., Kent, S. B. H. (1992) Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. Science 257, 1885–1894.PubMedCrossRefGoogle Scholar
  6. 6.
    Chait, B. T. (2006) Mass spectrometry: bottom-up or top-down. Science 314, 65–66.PubMedCrossRefGoogle Scholar
  7. 7.
    Veenstra, T. D., Martinovic, S., Anderson, G. A., Pasa-Tolic, L., Smith, R. D. (2000) Proteome analysis using selective incorporation of isotopically labeled amino acids. J Am Soc Mass Spectrom 11, 78–82.PubMedCrossRefGoogle Scholar
  8. 8.
    Du, Y., Parks, B. A., Sohn, S., Kwast, K. E., Kelleher, N. L. (2006) Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry. Anal Chem 78, 686–694.PubMedCrossRefGoogle Scholar
  9. 9.
    Lee, J. E., Kellie, J. F., Tipton, J. D., Catherman, A. D., Thomas, H. M., Ahlf, D. R., Durbin, K. R., Vellaichamy, A., Ntai, I., Marshall, A. G., Kelleher, N. L. (2009) A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome. J Am Soc Mass Spectrom 20, 2183–2191.PubMedCrossRefGoogle Scholar
  10. 10.
    Btancia, F. L., Montgomery, H., Tanaka, K., Kumashiro, S. (2004) Guanidino labeling derivatization strategy for global characterization of peptide mixtures by liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 76, 2748–2755.CrossRefGoogle Scholar
  11. 11.
    Ross, P. L., Huang, Y. M., 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. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3, 1154–1169.PubMedCrossRefGoogle Scholar
  12. 12.
    Schmidt, A., Kellermann, J., Lottspeich, F. (2005) A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5, 4–15.PubMedCrossRefGoogle Scholar
  13. 13.
    Yang, J., Gitlin, I., Krishnamurthy, V. M., Vazquez, J. A., Costello, C. E., Whitesides, G. M. (2003) Synthesis of monodisperse polymers from proteins. J Am Chem Soc 125, 12392–12393.PubMedCrossRefGoogle Scholar
  14. 14.
    Abello, N., Kerstjens, H. A. M., Postma, D. S., Bischoff, R. (2007) Selective acylation of primary amines in peptides and proteins. J Proteome Res 6, 4770–4776.PubMedCrossRefGoogle Scholar
  15. 15.
    Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A. J. R. (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4, 484–494.PubMedCrossRefGoogle Scholar
  16. 16.
    Fu, Q., Li, L. (2005) 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.PubMedCrossRefGoogle Scholar
  17. 17.
    Gidley, M. J., Sanders, J. K. (1982) Reductive methylation of proteins with sodium cyanoborohydride. Identification, suppression and possible uses of N-cyanomethyl by-products. Biochem J 224, 331–334.Google Scholar
  18. 18.
    Means G. E., Feeney R. E. (1995) Reductive alkylation of proteins. Anal Biochem 224, 1–16.PubMedCrossRefGoogle Scholar
  19. 19.
    Russo, A., Chandramouli, N., Zhang, L., Deng, H. (2008) Reductive glutaraldehydation of amine groups for identification of protein N-termini. J Proteome Res 7, 4178–4182.PubMedCrossRefGoogle Scholar
  20. 20.
    Goodlett, D. R., Keller, A., Watts, J. D., Newitt, R., Yi, E. C., Purvine, S., Eng, J. K., Von Haller, P., Aebersold, R., Kolker, E. (2001) Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation. Rapid Comm Mass Spectrom 15, 1214–1221.CrossRefGoogle Scholar
  21. 21.
    Ma, M., Kutz-Naber, K. K., Li, L. (2007) Methyl esterification assisted MALDI FTMS characterization of the orcokinin neuropeptide family. Anal Chem 79, 673–681.PubMedCrossRefGoogle Scholar
  22. 22.
    Fixarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F., White, F. M. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotech 20, 301–305.CrossRefGoogle Scholar
  23. 23.
    Houen, G., Svaerke, C., Barkholt, V. (1999) The solubilities of denatured proteins in different organic solvents. Acta Chem Scand 53, 1122–1126.PubMedCrossRefGoogle Scholar
  24. 24.
    Hoare, D. G., Koshland, D. E., Jr (1967) A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J Biol Chem 242, 2447–2453.PubMedGoogle Scholar
  25. 25.
    Sekiya, S., Wada, Y., Tanaka, K. (2004) Improvement of the MS/MS fragmentation ion coverage of acidic residue-containing peptides by amidation with 15N-substituted amine. Anal Chem 76, 5894–5902.PubMedCrossRefGoogle Scholar
  26. 26.
    Xu, Y., Zhang, L., Lu, H., Yang, P. (2008) Mass spectrometry analysis of phosphopeptides after peptide carboxy group derivatization. Anal Chem 80, 8324–8328.PubMedCrossRefGoogle Scholar
  27. 27.
    Jacobsen, J. R., Cochran, A. G., Stephans, J. C., King, D. S., Schultz, P. G. (1995) Mechanistic studies of anti-body-catalyzed pyrimidine dimmer photocleavage. J Am Chem Soc 117, 5453–5461.CrossRefGoogle Scholar
  28. 28.
    Lewis, W. G., Magallon, F. G., Fokin, V. V., Finn, M. G. (2004) Discovery and characterization of catalysts for azide-alkyne cycloaddition by fluorescence quenching. J Am Chem Soc 126, 9152–9153.PubMedCrossRefGoogle Scholar
  29. 29.
    Arnold, U., Ulbrich-Hofmann, R. (1999) Quantitative protein precipitation from guanidine hydrochloride-containing solutions by sodium deoxycholate/trichloroacetic acid. Anal Biochem 271, 197–199.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Casey J. Krusemark
    • 1
    • 2
    Email author
  • Brian L. Frey
    • 3
  • Lloyd M. Smith
    • 3
  • Peter J. Belshaw
    • 4
    • 5
  1. 1.Department of BiochemistryUniversity of WisconsinMadisonUSA
  2. 2.Stanford University BiochemistryStanfordUSA
  3. 3.Department of ChemistryUniversity of WisconsinMadisonUSA
  4. 4.Department of Chemistry and BiochemistryUniversity of WisconsinMadisonUSA
  5. 5.Best Sensors Inc.MiltonCanada

Personalised recommendations