Thermal unfolding of proteins probed by laser spray mass spectrometry.
The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.
Shi
Xiangguo
X
International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Takamizawa
Atsushi
A
Nishimura
Yoshifumi
Y
Hiraoka
Kenzo
K
Akashi
Satoko
S
D002021:Buffers; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry; D006358:Hot Temperature; D007202:Indicators and Reagents; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000737:chemistry
D002021:Buffers; D007202:Indicators and Reagents; D011506:Proteins; D025801:Ubiquitin; D045304:Cytochromes c