Gentle protein ionization assisted by high-velocity gas flow.
Gentle protein electrospray ionization is achieved using the high-velocity gas flow of an air amplifier to improve desolvation in conventional ESI and generate intact folded protein ions in the gas phase. Comparisons are made between the ESI spectra of a number of model proteins, including ubiquitin, cytochrome c, lysozyme, and myoglobin, over a range of pH values under optimized conditions, with and without using an air amplifier to achieve high-velocity gas flow. Previously reported increased ion signals are confirmed. In addition, the peaks recorded using the air amplifier are shown to be narrower, corresponding to more complete desolvation. Significant changes in the charge-state distribution also are observed, with a shift to lower charge state at high-velocity flow. The relationship between the observed charge-state distribution and protein conformation was explored by comparing the charge-state shifts and the distributions of charge states for proteins that are or are not stable in their native conformations in low pH solutions. The data suggest retention of native or nativelike protein conformations using the air amplifier in all cases examined. This is explained by a mechanism in which the air amplifier rapidly creates small droplets from the original large ESI droplets and these microdroplets then desolvate without a significant decrease in pH, resulting in retention of the folded protein conformations. Furthermore, the holoform of ionized myoglobin is visible at pH 3.5, a much lower value than the minimum needed to see this form in conventional ESI. These results provide evidence for the importance of the conditions used in the desolvation process for the preservation of the protein conformation and suggest that the conditions achieved when using high-velocity gas flows to assist droplet evaporation and ion desolvation are much gentler than those in conventional ESI experiments.
Yang
Pengxiang
P
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Cooks
R Graham
RG
Ouyang
Zheng
Z
Hawkridge
Adam M
AM
Muddiman
David C
DC
D045304:Cytochromes c / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry
D007477:Ions; D009211:Myoglobin; D011506:Proteins; D025801:Ubiquitin; D045304:Cytochromes c