Ray-Mediated Oxidative Labeling for Detecting Protein Conformational Changes by Electrospray Mass Spectrometry
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- 作者：Xin Tong ; J. Clara Wren ; Lars Konermann
- 刊名：Analytical Chemistry
- 出版年：2008
- 出版时间：March 15, 2008
- 年：2008
- 卷：80
- 期：6
- 页码：2222 - 2231
- 全文大小：289K
- 年卷期：v.80,no.6(March 15, 2008)
- ISSN：1520-6882
文摘
Exposure of proteins to hydroxyl radicals induces theincorporation of oxygen atoms into solvent-exposed sidechains. Earlier studies have employed this approach formapping protein-protein interactions in mass spectrometry-based footprinting experiments. This work exploreswhether the overall level of 
-ray mediated oxidativelabeling can be used for monitoring large-scale conformational changes. According to a recently developedkinetic model (Tong, X.; Wren, J. C.; Konermann, L.Anal. Chem. 2007, 79, 6376-6382), the apparent first-order rate constant for oxidative labeling can be approximated as kapp = kRAD/([P]tot + C/ku), where kRAD isthe primary rate of 
OH formation, [P]tot is the proteinconcentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant atwhich hydroxyl radicals react with the protein. The currentstudy introduces conformational effects into this modelby proposing that ku = 
journals/ancham/80/i06/eqn/ac702321re10001.gif">
ijournals/ancham/80/i06/eqn/ac702321re10002.gif">, where N is the numberof amino acids, i is a measure for the solvent exposureof residue i, and journals/ancham/80/i06/eqn/ac702321re10003.gif"> is the oxidation rate constant thatwould apply for a completely solvent-exposed side chain.Using myoglobin and cytochrome c as model systems, itis demonstrated that unfolding by addition of H3PO4increases kapp by up to 30% and 70%, respectively.Unfolding by other commonly used denaturants such asorganic acids or urea results in dramatically lower oxidation levels than for the native state, a behavior that is dueto the radical scavenging activity of these substances(corresponding to an increased value of C). Controlexperiments on model peptides are suitable for identifyingsuch "secondary" effects, i.e., factors that modify oxidation levels without being related to conformational changes.In conclusion, the overall OH labeling level represents aviable probe of large-scale protein conformational changesonly under conditions where secondary effects are knownto be minimal and where [P]tot is constant.
-ray mediated oxidativelabeling can be used for monitoring large-scale conformational changes. According to a recently developedkinetic model (Tong, X.; Wren, J. C.; Konermann, L.Anal. Chem. 2007, 79, 6376-6382), the apparent first-order rate constant for oxidative labeling can be approximated as kapp = kRAD/([P]tot + C/ku), where kRAD isthe primary rate of OH formation, [P]tot is the proteinconcentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant atwhich hydroxyl radicals react with the protein. The currentstudy introduces conformational effects into this modelby proposing that ku = ii is a measure for the solvent exposureof residue i, and OH labeling level represents aviable probe of large-scale protein conformational changesonly under conditions where secondary effects are knownto be minimal and where [P]tot is constant.