Secondary Reactions and Strategies To Improve Quantitative Protein Footprinting

摘要

Hydroxyl radical-mediated footprinting permits detailed examination of sthecture and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides aregoverned by changes in the solVent accessibility of the side-chain probe residues. The precise and accufatedetermination of peptide reaction rates is essential to successfully probing protein structure using footprinting.In this study, we specifically examine the magnitUde and mechanisms of secondary oxidation occurring after radiolvic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroalde andother oxidative species generated during radiolysis, signilicandr impacting the oxldation of Met and Cys but notaromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed byadding trace amounts of catalase or millimolar Met-NH_2 (or Met-OH) biber immediately after irradiahon; thisleads to greatly improved adherence to first-order kinetics and more precise observed oxidation fates. The strategyis shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disuilide as the principal product. The disulfides can be reduced before mass spectrometric analySis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.