Trypsin-Catalyzed N-Terminal Labeling of Peptides with Stable Isotope-Coded Affinity Tags for Proteome Analysis

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• 作者：Yanbo Pan ; Mingliang Ye ; Hao Zheng ; Kai Cheng ; Zhen Sun ; Fangjie Liu ; Jing Liu ; Keyun Wang ; Hongqiang Qin ; Hanfa Zou
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：January 21, 2014
• 年：2014
• 卷：86
• 期：2
• 页码：1170-1177
• 全文大小：437K
• ISSN：1520-6882

文摘

An enzymatic approach to label peptide N-termini with isotope-coded affinity tags is presented. This method exploits the high activity of trypsin for peptide synthesis in organic solvents. A cosubstrate containing a stable isotope-coded Arg residue and a biotin tag was synthesized. When the cosubstrate was incubated with tryptic peptides and trypsin in ethanol solution, the stable isotope-coded affinity tag was specifically coupled onto the N-termini of peptides via the formation of new peptide bonds. The labeled peptides were specifically enriched by avidin affinity chromatography and then were submitted to liquid chromatography鈥搕andem mass spectrometry (LC/MS/MS) for quantification. This enrichment step effectively reduced the interference by unlabeled peptides. The excellent performance of this approach was demonstrated by labeling standard peptides as well as a mouse liver digest. In addition to one amino acid residue, a few dipeptide tags were also introduced to the N-termini of peptides successfully by this enzymatic approach. It was found that the identifications for samples labeled with these tags were highly complementary. Coupling a short sequence tag onto peptides could be an effective approach to improve the coverage for proteome analysis.