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Diethylpyrocarbonate labeling for the structural analysis of proteins: label scrambling in solution and how to avoid it.


ABSTRACT: Covalent labeling along with mass spectrometry is a method that is increasingly used to study protein structure. Recently, it has been shown that diethylpyrocarbonate (DEPC) is a powerful labeling reagent because it can modify up to 30% of the residues in the average protein, including the N-terminus, His, Lys, Tyr, Ser, Thr, and Cys residues. We recently discovered, however, that Cys residues that form disulfide bonds appear to be modified by DEPC as well. In this work, we demonstrate that disulfide linked Cys residues are not actually reactive with DEPC but, instead, once reduced, free Cys residues can capture a carbethoxy group from other modified amino acids via a solution-phase reaction that can occur during the protein digestion step. This "scrambling" of carbethoxy groups decreases the amount of modification observed at other residues and can potentially provide incorrect protein structural information. Fortunately, label scrambling can be completely avoided by alkylating the free thiols after disulfide reduction.

SUBMITTER: Zhou Y 

PROVIDER: S-EPMC3324597 | BioStudies | 2012-01-01

REPOSITORIES: biostudies

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