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Rapid identification of oxidation‐induced conformational changes by kinetic analysis
Author(s) -
Venkatesh Sanjay,
Tomer Kenneth B.,
Sharp Joshua S.
Publication year - 2007
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3291
Subject(s) - chemistry , kinetics , oxygen , mass spectrometry , derivatization , reactive oxygen species , redox , protein structure , reagent , biophysics , chromatography , biochemistry , organic chemistry , physics , quantum mechanics , biology
Abstract Protein oxidation by reactive oxygen species is known to result in changes in the structure and function of the oxidized protein. Many proteins can tolerate multiple oxidation events before altering their conformation, while others suffer gross changes in conformation after a single oxidation event. Additionally, reactive oxygen species have been used in conjunction with mass spectrometry to map the accessible surface of proteins, often after verification that the oxidations do not alter the conformation. However, detection of oxidation‐induced conformational changes by detailed kinetic oxidation analysis of individual proteolytic peptides or non‐mass spectrometric analysis is labor‐intensive and often requires significant amounts of sample. In this work, we describe a methodology to detect oxidation‐induced conformational changes in proteins via direct analysis of the intact protein. The kinetics of addition of oxygen to unmodified protein are compared with the kinetics of addition of oxygen to the mono‐oxidized protein. These changes in the rate of oxidation of the oxidized versus the non‐oxidized protein are strongly correlated with increases in the random coil content as measured by the molar ellipticity at 198 nm. This methodology requires only small amounts of protein, and can be done rapidly without additional sample handling or derivatization. Copyright © 2007 John Wiley & Sons, Ltd.