title: Site-specific proteomics approach for study protein S-nitrosylation.
abstract: Here we present a novel and robust method for the identification of protein S-nitrosylation sites in complex protein mixtures. The approach utilizes the cysteinyl affinity resin to selectively enrich S-nitrosylated peptides reduced by ascorbate followed by nanoscale liquid chromatography tandem mass spectrometry. Two alkylation agents with different added masses were employed to differentiate the S-nitrosylation sites from the non-S-nitrosylation sites. We applied this approach to MDA-MB-231 cells treated with Angeli's salt, a nitric oxide donor that has been shown to inhibit breast tumor growth and angiogenesis. A total of 162 S-nitrosylation sites were identified and an S-nitrosylation motif was revealed in our study. The 162 sites are significantly more than the number reported by previous methods, demonstrating the efficiency of our approach. Our approach will further facilitate the functional study of protein S-nitrosylation in cellular processes and may reveal new therapeutic targets.
authors.affiliation: Department of Pathology and Microbiology, Mass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
authors.forename: Miao
authors.initials: M
authors.lastname: Liu
authors.forename: Jinxuan
authors.initials: J
authors.lastname: Hou
authors.forename: Lin
authors.initials: L
authors.lastname: Huang
authors.forename: Xin
authors.initials: X
authors.lastname: Huang
authors.forename: Tyler H
authors.initials: TH
authors.lastname: Heibeck
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Pasa-Tolic
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Kai
authors.initials: K
authors.lastname: Fu
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Zhang
authors.forename: Steven H
authors.initials: SH
authors.lastname: Hinrichs
authors.forename: Shi-Jian
authors.initials: SJ
authors.lastname: Ding
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry; D001665:Binding Sites; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D011506:Proteins; D009569:Nitric Oxide; D014357:Trypsin; D003545:Cysteine; D001205:Ascorbic Acid