Resin-Assisted Enrichment of N-Terminal Peptides for Characterizing Proteolytic Processing
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- 作者：Jong-Seo Kim ; Ziyu Dai ; Uma K. Aryal ; Ronald J. Moore ; David G. Camp ; II ; Scott E. Baker ; Richard D. Smith ; Wei-Jun Qian
- 刊名：Analytical Chemistry
- 出版年：2013
- 出版时间：July 16, 2013
- 年：2013
- 卷：85
- 期：14
- 页码：6826-6832
- 全文大小：319K
- 年卷期：v.85,no.14(July 16, 2013)
- ISSN：1520-6882
文摘
A resin-assisted enrichment method has been developed for specific isolation of protein N-terminal peptides to facilitate LC-MS/MS characterization of proteolytic processing, a major form of posttranslational modifications. In this method, protein thiols are blocked by reduction and alkylation, and protein lysine residues are converted to homoarginines. Protein N-termini are selectively converted to reactive thiol groups, and the thiol-containing N-terminal peptides are then captured by a thiol-affinity resin with high specificity (>97%). The efficiencies of these sequential reactions were demonstrated to be nearly quantitative. The resin-assisted N-terminal peptide enrichment approach was initially applied to a cell lysate of the filamentous fungus Aspergillus niger. Subsequent C-MS/MS analyses resulted in the identification of 1672 unique protein N-termini or proteolytic cleavage sites from 690 unique proteins.