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Desulfurization of cysteine‐containing peptides resulting from sample preparation for protein characterization by mass spectrometry
Author(s) -
Wang Zhouxi,
Rejtar Tomas,
Zhou Zhaohui Sunny,
Karger Barry L.
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4383
Subject(s) - dehydroalanine , chemistry , tcep , cysteine , mass spectrometry , chromatography , alanine , combinatorial chemistry , phosphine , amino acid , organic chemistry , biochemistry , catalysis , enzyme
In this study, we have examined two cysteine modifications resulting from sample preparation for protein characterization by mass spectrometry (MS): (1) a previously observed conversion of cysteine into dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine into alanine. Using model peptides, the conversion of cysteine into dehydroalanine via β ‐elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37°C, pH 7.0–9.0) without disulfide reduction and alkylation. Furthermore, the surprising conversion of cysteine into alanine was shown to occur by heating cysteine‐containing peptides in the presence of a phosphine (tris(2‐carboxyethyl)phosphine hydrochloride (TCEP)). The formation of alanine from cysteine, investigated by performing experiments in H 2 O or D 2 O, suggested a radical‐based desulfurization mechanism unrelated to β ‐elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. Copyright © 2010 John Wiley & Sons, Ltd.