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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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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