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.
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References
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.
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.
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.
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.
Chait, B. T., Kent, S. B. H. (1992) Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. Science 257, 1885–1894.
Chait, B. T. (2006) Mass spectrometry: bottom-up or top-down. Science 314, 65–66.
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.
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.
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.
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.
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.
Schmidt, A., Kellermann, J., Lottspeich, F. (2005) A novel strategy for quantitative proteomics using isotope-coded protein labels. Proteomics 5, 4–15.
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.
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.
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.
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.
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.
Means G. E., Feeney R. E. (1995) Reductive alkylation of proteins. Anal Biochem 224, 1–16.
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.
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.
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.
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.
Houen, G., Svaerke, C., Barkholt, V. (1999) The solubilities of denatured proteins in different organic solvents. Acta Chem Scand 53, 1122–1126.
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.
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.
Xu, Y., Zhang, L., Lu, H., Yang, P. (2008) Mass spectrometry analysis of phosphopeptides after peptide carboxy group derivatization. Anal Chem 80, 8324–8328.
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.
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.
Arnold, U., Ulbrich-Hofmann, R. (1999) Quantitative protein precipitation from guanidine hydrochloride-containing solutions by sodium deoxycholate/trichloroacetic acid. Anal Biochem 271, 197–199.
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.
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Krusemark, C.J., Frey, B.L., Smith, L.M., Belshaw, P.J. (2011). Complete Chemical Modification of Amine and Acid Functional Groups of Peptides and Small Proteins. In: Gevaert, K., Vandekerckhove, J. (eds) Gel-Free Proteomics. Methods in Molecular Biology, vol 753. Humana Press. https://doi.org/10.1007/978-1-61779-148-2_6
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DOI: https://doi.org/10.1007/978-1-61779-148-2_6
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