title: Trypsin-catalyzed N-terminal labeling of peptides with stable isotope-coded affinity tags for proteome analysis.
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.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Yanbo
authors.initials: Y
authors.lastname: Pan
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Hao
authors.initials: H
authors.lastname: Zheng
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Zhen
authors.initials: Z
authors.lastname: Sun
authors.forename: Fangjie
authors.initials: F
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Keyun
authors.initials: K
authors.lastname: Wang
authors.forename: Hongqiang
authors.initials: H
authors.lastname: Qin
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002247:Carbon Isotopes; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D005260:Female; D008099:Liver / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D002247:Carbon Isotopes; D010455:Peptides; D020543:Proteome; D001360:Avidin; D001710:Biotin; D014357:Trypsin