1. Trypsin-Catalyzed N-Terminal Labeling of Peptides with Stable Isotope-Coded Affinity Tags for Proteome Analysis
- Author
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Jing Liu, Mingliang Ye, Kai Cheng, Fangjie Liu, Hongqiang Qin, Yanbo Pan, Keyun Wang, Hanfa Zou, Hao Zheng, and Zhen Sun
- Subjects
Proteome ,Molecular Sequence Data ,Biotin ,Peptide ,Tandem mass tag ,Chromatography, Affinity ,Analytical Chemistry ,Mice ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Peptide synthesis ,medicine ,Animals ,Trypsin ,Amino Acid Sequence ,chemistry.chemical_classification ,Carbon Isotopes ,Dipeptide ,Chromatography ,Staining and Labeling ,biology ,Avidin ,Isotope-coded affinity tag ,Mice, Inbred C57BL ,Isobaric labeling ,Liver ,chemistry ,Biochemistry ,Proteolysis ,biology.protein ,Female ,Peptides ,Chromatography, Liquid ,medicine.drug - Abstract
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-tandem 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.
- Published
- 2014
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