Cysteine-Selective Peptide Identification: Selenium-Based Chromophore for Selective S-Se Bond Cleavage with 266 nm Ultraviolet Photodissociation.
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.
Department of Chemistry, University of Texas , Austin, Texas 78712, United States.
W Ryan
WR
Parker
Department of Chemistry, University of Texas , Austin, Texas 78712, United States.
Dustin D
DD
Holden
Department of Chemistry, University of Texas , Austin, Texas 78712, United States.
Victoria C
VC
Cotham
Department of Chemistry, University of Texas , Austin, Texas 78712, United States.
Hua
H
Xu
Department of Chemistry, University of Texas , Austin, Texas 78712, United States.
Jennifer S
JS
Brodbelt
D000477:Alkylating Agents / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D010782:Photolysis / Q000528:radiation effects; D010797:Phthalimides / Q000737:chemistry; D012643:Selenium / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
D000477:Alkylating Agents; D010455:Peptides; D010797:Phthalimides; D013438:Sulfhydryl Compounds; C037431:phthalimide; D012710:Serum Albumin, Bovine; D045304:Cytochromes c; D012643:Selenium; D003545:Cysteine