Secondary Reactions and Strategies To Improve Quantitative Protein Footprinting

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• 作者：Guozhong Xu ; Janna Kiselar ; Qin He ; and Mark R. Chance
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：May 15, 2005
• 年：2005
• 卷：77
• 期：10
• 页码：3029 - 3037
• 全文大小：176K
• 年卷期：v.77,no.10(May 15, 2005)
• ISSN：1520-6882

文摘

Hydroxyl radical-mediated footprinting permits detailedexamination of structure and dynamic processes of proteins and large biological assemblies, as changes in therate of reaction of radicals with target peptides aregoverned by changes in the solvent accessibility of theside-chain probe residues. The precise and accuratedetermination of peptide reaction rates is essential tosuccessfully probing protein structure using footprinting.In this study, we specifically examine the magnitude andmechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis.Secondary oxidation results from hydrogen peroxide andother oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but notaromatic or other reactive residues. Secondary oxidationof Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibilityof Met-containing peptides. It can be suppressed byadding trace amounts of catalase or millimolar Met-NH₂(or Met-OH) buffer immediately after irradiation; thisleads to greatly improved adherence to first-order kineticsand more precise observed oxidation rates. The strategyis shown to suppress secondary oxidation in modelpeptides and improve data quality in examining thereactivity of peptides within the Arp2/3 protein complex.Cysteine is also subject to secondary oxidation generatingdisulfide as the principal product. The disulfides can bereduced before mass spectrometric analysis by reducingagents such as TCEP, while methionine sulfoxide isrefractory to reduction by this reagent under typicalreducing conditions.