title: Resin-assisted enrichment of N-terminal peptides for characterizing proteolytic processing.
abstract: 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.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.
authors.forename: Jong-Seo
authors.initials: JS
authors.lastname: Kim
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Dai
authors.forename: Uma K
authors.initials: UK
authors.lastname: Aryal
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Scott E
authors.initials: SE
authors.lastname: Baker
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D007475:Ion Exchange Resins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism*; D059748:Proteolysis; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007475:Ion Exchange Resins; D010446:Peptide Fragments