title: Mass spectrometry-based sequencing of protein C-terminal peptide using α-carboxyl group-specific derivatization and COOH capturing.
abstract: An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.
authors: Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Kyoto 604-8511, Japan.
authors: Chihiro
authors: C
authors: Nakajima
authors: Hiroki
authors: H
authors: Kuyama
authors: Koichi
authors: K
authors: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000032:analysis / Q000737:chemistry; D019004:Galanin / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D011437:Propylamines / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*; D013482:Superoxide Dismutase / Q000032:analysis / Q000737:chemistry
chemical_list: D002147:Calmodulin; D010455:Peptides; D011437:Propylamines; D011506:Proteins; D019004:Galanin; D013482:Superoxide Dismutase