title: Targeted protein capture for analysis of electrophile-protein adducts.
abstract: Proteomic analyses of protein-electrophile adducts generally employ affinity capture of the adduct moiety, which enables global analyses, but is poorly suited to targeted studies of specific proteins. We describe a targeted molecular probe approach to study modifications of the molecular chaperone heat-shock protein 90 (Hsp90), which regulates diverse client proteins. Noncovalent affinity capture with a biotinyl analog of the HSP90 inhibitor geldanamycin enables detection of the native protein isoforms Hsp90α and Hsp90β and their phosphorylated forms. We applied this probe to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. This approach was also applied to measure the kinetics of site-specific adduction of selected Hsp90 residues. A protein-selective affinity capture approach is broadly applicable for targeted analysis of electrophile adducts and their biological effects.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Connor
authors.forename: Simona G
authors.initials: SG
authors.lastname: Codreanu
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D000447:Aldehydes / Q000737:chemistry; D016227:Benzoquinones / Q000737:chemistry; D015153:Blotting, Western; D045744:Cell Line, Tumor; D030562:Databases, Protein; D004583:Electrons; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000737:chemistry; D013058:Mass Spectrometry; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D000447:Aldehydes; D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D014357:Trypsin; C027576:4-hydroxy-2-nonenal; C001277:geldanamycin