Abstract
The analysis of protein modification by electrophiles is a challenging problem. Most reported protein-electrophile adducts have been characterized from in vitro reactions or through affinity capture of the adduct moiety, which enables global analyses but is poorly suited to targeted studies of specific proteins. We employed 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-geldanamycin probe isolated both isoforms of the native protein (Hsp90α and Hsp90β) from human RKO colorectal cancer cells. Geldanamycin-biotin capture afforded higher purity Hsp90 than did immunoprecipitation and enabled detection of endogenously phosphorylated protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We applied this approach to map and quantify adducts formed on Hsp90 by 4-hydroxynonenal (HNE) in RKO cells. LC-MS/MS analyses of tryptic digests by identified His(450) and His(490) of Hsp90α as having a 158 Da modification, corresponding to NaBH(4)-reduced HNE adducts. Five histidine residues were also adducted on Hsp90β: His(171), His(442), His(458), His(625), and His(632). The rates of adduction at these sites were determined with Hsp90 protein in vitro and with Hsp90 in HNE-treated cells with a LC-MS/MS-based, label-free relative quantitation method. During in vitro and cell treatment with HNE, residues on Hsp90α and Hsp90β displayed adduction rates ranging from 3.0 × 10(-5) h(-1) to 1.08 ± 0.17 h(-1). Within the middle client-binding domain of Hsp90α, residue His(450) demonstrated the most rapid adduction with k(obs) of 1.08 ± 0.17 h(-1) in HNE-treated cells. The homologous residue on Hsp90β, His(442), was adducted more rapidly than the N-terminal residue, His(171), despite very similar predicted pK(a) values of both residues. The Hsp90 middle client-binding domain thus may play a signicant role in HNE-mediated disruption of Hsp90-client protein interactions. The results illustrate the utility of a protein-selective affinity capture approach for targeted analysis of electrophile adducts and their biological effects.