Supercharging and multiple reaction monitoring of high-molecular-weight intact proteins using triple quadrupole mass spectrometry.

Abstract

Different supercharging agents were tested to study their effect on the intensity and charge state distributions of high-molecular-weight intact proteins. The goal of this work was to increase chargeability and ionization efficiency for proteins ranging from 66 to 150 kDa, to enable subsequent optimization of multiple reaction monitoring (MRM) mode transitions with a triple quadrupole mass spectrometer for potential top-down quantitative analysis. Supercharging agents, such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol (TFE), and sulfolane were tested in different concentrations in 50/50 acetonitrile/water with 0.5% formic acid to examine the electrospray ionization response for three model proteins: bovine serum albumin (66 kDa), holo-transferrin (78 kDa), and immunoglobulin G (150 kDa). The settings of ionization source temperature and mobile phase flow rate were also examined. MRM transitions were developed for a wide range of precursor ions for each protein, and limits of detection were determined for the proteins in the presence of favorable additive combinations. For most of the proteins, m-NBA (1%) and TFE (5%) worked most effectively, both to shift the charge state and increase intensity. This is the first report of the use of TFE as an effective agent for both increasing protein chargeability and ionization response. TFE increased ionization efficiency between 3- and 14-fold for the model proteins studied. Increases in both source temperature and flow rate reduced the magnitude of the average charge state observed. The MRM transitions of six to eight different precursor ions of the proteins were optimized and limits of detection in the nanogram quantity on column were determined. The feasibility for top-down quantitative analysis of high-molecular-weight proteins with a triple quadrupole mass spectrometer was demonstrated. Further, additives such as TFE can be highly beneficial for increased chargeability and response of the proteins.