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

Abstract

Rationale: 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. Methods: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.Results: 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. Conclusions:The feasibility for top-down quantitative analysis of high-molecularweight 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.

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

“…When optimizing MRM transitions, in contrast with their optimization for small molecules where usually a complete fragmentation of precursor ion is preferred to gain signal intensity, for intact proteins, reproducible and unique product ions are usually generated at lower collision energy (more selective fragmentations at the weakest links of the protein), where the precursor ion still remains prominent in the spectra [14,15,25]. The optimal collision energy depends also on the molecular weight of a protein and its charge state.…”

“…The MRM summation of the responses of different ions has been shown to potentially improve the sensitivity of low abundance proteins and to improve the LOD [15,25,40,41]. The efficiency of these techniques are often analyte dependent and consequently, whether summation of ion intensities is beneficial needs to be determined case by case depending on the actual m / z values, extraction windows used, and the presence of matrix interferences.…”

“…DFA effectively lowers pH to suppress deleterious silanol interactions; it is also a strong ion‐pairing agent suitable to improve RP‐LC separations, but it does not decrease MS sensitivity as much as TFA. Several studies have evaluated the effect of mobile phase additives, with a focus on TFA, for the analysis of peptides and proteins [22–25], but only one recent study dealt specifically with the use of DFA for improving LC–MS characterization of antibody–drug conjugates [21]. The authors have shown that it was possible to increase MS sensitivity two to three times and to improve the recovery as well as chromatographic resolution of subunit‐digested monoclonal antibody by replacing TFA with DFA as a mobile phase modifier.…”

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…While the vast majority of top‐down proteomic analyses have been performed with HRMS instrumentation, some recent work has shown the potential for use of less expensive and more prevalent, but low resolution, QQQ‐MS instruments [7,27,65,98,99]. Used in selected or multiple reaction monitoring (SRM and MRM, respectively) mode, the QQQ‐MS is the gold standard for trace quantitative analysis of small molecules from biological fluids.…”

“…Khanal et al. [99] investigated much larger proteins; they were still able to reach detection limits in the 10 to 25 μg/mL range using different additives to aid ionization and supercharging for bovine serum albumin (BSA; 66 kDa), human transferrin (78 kDa), and an IgG antibody (150 kDa). Of note, in this latter work, the addition of 1 – 5% 2,2,2‐trifluoroethanol to the spray solvent was shown to provide substantial increases in protein charging and ionization efficiency for the intact antibody protein.…”