Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions

Abstract

In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems.

Dissecting the structural heterogeneity of proteins by native mass spectrometry

“…As the molecular weight increases, individual glycoforms become harder to resolve. Native top‐down is one solution to dissect the intact complexes into smaller constituents that can be better resolved by MS, and orthogonal separation techniques (Cramer et al, 2022; Liu et al, 2020; Shen et al, 2018) are also essential to reduce the heterogeneity before MS. Other methods to deal with heterogeneity utilize gas‐phase chemistry to further fractionate the subpopulations, or dilute the heterogeneity by increasing their separation in m/z space (Chen, Dai, et al, 2022; Wang et al, 2017; Yang et al, 2021). While many of these methods are still in the research stage, they will undoubtedly become more accessible as the techniques mature.…”

Mass Spectrometry in China

“…Guo et al demonstrated the use of tandem MS fragments in predicting degradation products of metabolites in drug discovery . Wang et al utilized gas-phase reactions for limited charge reduction of heterogeneous intact glycoproteins that were otherwise not resolvable with direct MS analysis . Stereochemistry is important in medicine and neurological diseases such as Alzheimer’s disease.…”

“…16 Wang et al utilized gas-phase reactions for limited charge reduction of heterogeneous intact glycoproteins that were otherwise not resolvable with direct MS analysis. 17 chemistry is important in medicine and neurological diseases such as Alzheimer's disease. Yet differentiation of isomers is challenging by MS alone.…”

Mass spectrometry‐based methods to characterize highly heterogeneous biopharmaceuticals, vaccines, and nonbiological complex drugs at the intact‐mass level

“…A similar approach was recently used by Wang and colleagues to perform meaningful mass measurements on intact glycoproteins with the carbohydrate content exceeding 20% by weight (Q. Chen et al, 2022). Moving beyond the realm of proteins, a similar approach was used recently to obtain meaningful information on the mass distributions of oligomeric species within Tween R 80 (Betancourt et al, 2016).…”

“…More recently, the original proton‐transfer scheme (McLuckey & Goeringer, 1995) was reintroduced by Bush and coworkers (Laszlo & Bush, 2015) who used even‐electron anions as proton acceptors to induce a controlled reduction of the number of charges accommodated by the analyte (polycationic) species in the gas phase. A similar approach was recently used by Wang and colleagues to perform meaningful mass measurements on intact glycoproteins with the carbohydrate content exceeding 20% by weight (Q. Chen et al, 2022). Moving beyond the realm of proteins, a similar approach was used recently to obtain meaningful information on the mass distributions of oligomeric species within Tween R 80 (Betancourt et al, 2016).…”