Journal
ANALYTICAL BIOCHEMISTRY
Volume 428, Issue 2, Pages 167-172Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.06.016
Keywords
C-terminal sequencing; alpha-COOH-specific derivatization; COOH capture
Funding
- Japan Society for the Promotion of Science (JSPS)
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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 alpha-carboxyl group (alpha-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of alpha-COOH was achieved by a combination of specific activation of alpha-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. (C) 2012 Elsevier Inc. All rights reserved.
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