Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry.
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 beta-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37 degrees 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 beta-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.
Wang
Zhouxi
Z
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
Rejtar
Tomas
T
Zhou
Zhaohui Sunny
ZS
Karger
Barry L
BL
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D010455:Peptides; D011506:Proteins; D003545:Cysteine; D000409:Alanine