Hydroxyl Radical-Mediated Modification of Proteins as Probes for Structural Proteomics

Structural Analysis of Proinsulin Hexamer Assembly by Hydroxyl Radical Footprinting and Computational Modeling

“…The largest changes from monomer to hexamer were observed within peptides B11-B22: in particular, residues Tyr B16 , Leu B17 , and Arg B22 exhibited a 4.8-fold decrease in oxidation rate. These observations suggest that the latter reactive side chains are protected in the HI hexamer relative to HI monomer (8,12,22,23) , and Tyr A19 in the A1-A21 segment. Although SAs for the rest of the oxidized side chains within peptides A1-A21 exhibited no changes upon hexamer formation, the SA for the Leu A13 side chain (as calculated from the crystal structure) decreased from 44 Å 2 (monomer) to 0.7 Å 2 (hexamer).…”

“…In particular, rates of oxidation of the side chains of Phe B24 , Phe B25 and Tyr B26 were attenuated by ϳ1.5-fold relative to HI; oxidation for Pro B28 was decreased by 2-fold. Because of the correlation between SA and reaction rates (8,12,22,23), these findings suggest that reactive side chains in peptides B23-B29 are more buried (experienced greatest decrease in SA) in HPI than in HI. These overall results are consistent with the NMR structure of DKP-proinsulin as an engineered monomer (10).…”

The Utility of Mass Spectrometry Based Structural Proteomics in Biopharmaceutical Biologics Development

“…The interpretation of high volumes of data resulting from covalent labeling experiments used to pose as the biggest bottleneck for the overall experiment, thus, limiting their potential. A typical hydroxyl radical-mediated covalent labeling experiment leads to multiple oxidation states of various amino acid side chains Takamoto & Chance (2006); Xu & Chance (2007), leading to a challenging task for data analysis. This bottleneck has now been eliminated with the advent of ProtMapMS, a computational analytical tool, that is specifically tailored to meet the needs of covalent labelling experiments Kaur et al (2009).…”

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting

“…Hydroxyl radical footprinting is one of the commonly used covalent labeling techniques. 15,37,39 Hydroxyl radicals can label up to 16 of 20 amino acid side chains and offer excellent sequence coverage. The structural information obtained can be quite detailed and rich, but the approach is impractical for many investigators because special equipment for generation of radicals is usually required.…”