Irreversible thermal denaturation of cytochrome C studied by electrospray mass spectrometry.
This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solution-phase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11+, whereas a considerably lower degree of oxidation is observed for the 7+ and 8+ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high- and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein.
Liu
Jiangjiang
J
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
Konermann
Lars
L
D000818:Animals; D002417:Cattle; D003198:Computer Simulation; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D006358:Hot Temperature; D009206:Myocardium / Q000737:chemistry; D016011:Normal Distribution; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism
D045304:Cytochromes c; D014357:Trypsin