ArgC-Like Digestion: Complementary or Alternative to Tryptic Digestion?

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• 作者：Vahid GolghalyaniMoritz Neupärtl ; Ilka Wittig ; Ute Bahr ; Michael Karas
• 刊名：Journal of Proteome Research
• 出版年：2017
• 出版时间：February 3, 2017
• 年：2017
• 卷：16
• 期：2
• 页码：978-987
• 全文大小：556K
• ISSN：1535-3907

文摘

Enzymatic digestion of complex protein samples is often performed by use of multiple proteases to improve protein identification and characterization. Combining trypsin with ArgC is one option to enhance sequence coverage in bottom-up proteomics. However, the low selectivity of this endoprotease derogates from the benefit of the combination. Our approach here is to mimic ArgC digestion by chemically modifying all lysine residues in proteins so that trypsin can only cleave C-terminal to arginine. Four different amine modifications, dimethylation, acetylation, propionylation, and carbethoxylation, were tested, and the protocols were optimized. A nearly complete conversion of the primary amines was achieved for all modifications. Tryptic digestion of Escherichia coli lysate proteins after acylation of lysine residues shows the most significant improvement compared with data received from ArgC digest. After propionylation, 9216 unique peptides identified 1439 proteins, which, compared with a conventional tryptic digestion, represents the identification of 150 additional proteins due to a reasonable reduction of the sample complexity and higher fragmentation efficiencies of the peptides. It is therefore concluded that the Arg-C like digestion should no longer be regarded as a complementary approach but forms a viable and superior alternative to the conventional trypsin digestion.