Cysteine-Selective Peptide Identification: Selenium-Based Chromophore for Selective S–Se Bond Cleavage with 266 nm Ultraviolet Photodissociation

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• 作者：W. Ryan Parker ; Dustin D. Holden ; Victoria C. Cotham ; Hua Xu ; Jennifer S. Brodbelt
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：July 19, 2016
• 年：2016
• 卷：88
• 期：14
• 页码：7222-7229
• 全文大小：445K
• 年卷期：0
• ISSN：1520-6882

文摘

The tremendous number of peptides identified in current bottom-up mass spectrometric workflows, although impressive for high-throughput proteomics, results in little selectivity for more targeted applications. We describe a strategy for cysteine-selective proteomics based on a tagging method that installs a S–Se bond in peptides that is cleavable upon 266 nm ultraviolet photodissociation (UVPD). The alkylating reagent, N-(phenylseleno)phthalimide (NPSP), reacts with free thiols in cysteine residues and attaches a chromogenic benzeneselenol (SePh) group. Upon irradiation of tagged peptides with 266 nm photons, the S–Se bond is selectively cleaved, releasing a benzeneselenol moiety corresponding to a neutral loss of 156 Da per cysteine. Herein we demonstrate a new MS/MS scan mode, UVPDnLossCID, which facilitates selective screening of cysteine-containing peptides. A “prescreening” event occurs by activation of the top N peptide ions by 266 nm UVPD. Peptides exhibiting a neutral loss corresponding to one or more SePh groups are reactivated and sequenced by CID. Because of the low frequency of cysteine in the proteome, unique cysteine-containing peptides may serve as surrogates for entire proteins. UVPDnLossCID does not generate as many peptide spectrum matches (PSMs) as conventional bottom-up methods; however, UVPDnLossCID provides far greater selectivity.