Charging of Proteins in Native Mass Spectrometry

Abstract

Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton-transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformation of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton-transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism.

Conditions for Analysis of Native Protein Structures Using Uniform Field Drift Tube Ion Mobility Mass Spectrometry and Characterization of Stable Calibrants for TWIM-MS

“…This subtle difference is consistent with a greater percentage of folded cytochrome c present under the conditions reported here. This charge state distribution is in agreement with that predicted by the Rayleigh limit (Z R ) for cytochrome c (8+) supporting that the protein detected is in a globular (folded) form [38].…”

Effects of aprotic solvents on the stability of metal‐free superoxide dismutase probed by native electrospray ionization–ion mobility–mass spectrometry

“…The similar trends of the charging (Figure S1) and Di/Mo ratio (Figure S2) of apo‐SOD1 were also observed in the presence of DMI and DMSO, but not for ACN and THF. Considering the ongoing debate on the mechanism of ESI, the charging and dissociation differences of apo‐SOD1 with three solvents may have resulted from many factors, including the different boiling points and the gas‐phase proton transfer reactions, solvent acidity and basicity . Moreover, we found that these three aprotic solvents can also cause signal broadening (Figure S3) that represents the formation of solvent adducts.…”

“…Considering the ongoing debate on the mechanism of ESI, the charging and dissociation differences of apo-SOD1 with three solvents may have resulted from many factors, including the different boiling points and the gas-phase proton transfer reactions, solvent acidity and basicity. [32][33][34] Moreover, we found that these three aprotic solvents can also cause signal broadening ( Figure S3) that represents the formation of solvent adducts. This finding is similar with previous reports on DMSO, 16,17 which can protect against the dissociation of protein by reducing coulomb repulsions due to the low charge state and cooling effect of adduct dissociation.…”

Interaction analysis of glycoengineered antibodies with CD16a: a native mass spectrometry approach

“…22 ESI produces multiply-charged signals with a narrow distribution of lower charge states if proteins are analyzed in "native" buffered aqueous solutions. 23 Using lowresolution MS, a nonglycosylated and slightly glycosylated proteins can be easily measured, whereas highly heterogeneous glycoproteins pose an analytical challenge due to overlapping ion signals from naturally existing glycoforms. 24 This limitation was highlighted by Wang et al 25 when determining the molecular weight (MW) of epidermal growth factor receptor and CD38.…”