The specific isolation of C‐terminal peptides of proteins through a transamination reaction and its advantage for introducing functional groups into the peptide |
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| Authors: | Kazuhiro Sonomura Hiroki Kuyama Ei‐ichi Matsuo Susumu Tsunasawa Osamu Nishimura |
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| Institution: | 1. Institute for Protein Research, Osaka University, Suita 565‐0871, Japan;2. Life Science Research Center, Technology Research Laboratory, Shimadzu Corporation, Kyoto 604‐8511, Japan |
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| Abstract: | A novel method for isolating C‐terminal peptides from proteolytic digests of proteins was developed. Proteins were digested with lysyl endopeptidase (LysC) and applied to metal‐ion‐catalyzed transamination reactions. This reaction enabled the selective conversion of an Nα‐amino group to a carbonyl group. Subsequent incubation with p‐phenylenediisothiocyanate (DITC) glass effectively scavenged the lysine‐containing N‐terminus and internal peptides. The obtained C‐terminal peptide is open to modification with reagents having virtually any type of functionality owing to the reactive α‐ketocarbonyl group. In this report, 2,4‐dinitrophenylhydrazine (DNPH) was used as an example of a nucleophile to the carbonyl group. The isolated C‐terminal peptide was modified with DNPH, which exhibited signal enhancement, and was sequenced by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Copyright © 2009 John Wiley & Sons, Ltd. |
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