Electrothermal supercharging of proteins in native electrospray ionization.

ABSTRACT: The formation of high charge-state protein ions with nanoelectrospray ionization (nESI) from purely aqueous ammonium bicarbonate solutions at neutral pH, where the proteins have native or native-like conformations prior to ESI droplet formation, is demonstrated. This "electrothermal" supercharging method depends on the temperature of the instrument entrance capillary, the nESI spray potential, and the solution ionic strength and buffer, although other factors almost certainly contribute. Mass spectra obtained with electrothermal supercharging appear similar to those obtained from denaturing solutions where charging beyond the total number of basic sites can be achieved. For example, a 17+ ion of bovine ubiquitin was formed by nESI of a 100 mM ammonium bicarbonate, pH 7.0, solution, which is three more charges than the total number of basic amino acids plus the N-terminus. Heating of the ESI droplets in the vacuum/atmosphere interface and the concomitant denaturation of the protein in the ESI droplets prior to ion formation appears to be the primary origin of the very high charge-state ions formed from these purely aqueous, buffered solutions. nESI mass spectra resembling those obtained under traditional native or denaturing conditions can be reversibly obtained simply by toggling the spray voltage between low and high values.

SUBMITTER: Sterling HJ 

PROVIDER: S-EPMC3328611 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature