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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-literature | 2012 May

REPOSITORIES: biostudies-literature

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

Zhou Yuping Y Vachet Richard W RW

Journal of the American Society for Mass Spectrometry 20120214 5

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 disu ...[more]

PMID: 22351293

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Project description:Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for ?-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for cytochrome c, and 9 to 8 Å for ?-2-microglobulin.

Dataset Information

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

Publications

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

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