To 200,000 m/z and beyond: native electron capture charge reduction mass spectrometry deconvolves heterogeneous signals in large biopharmaceutical analytes

Abstract

Great progress has been made in the detection of large biomolecular analytes by native mass spectro-metry, however characterizing highly heterogeneous samples remains challenging due to the presence of many overlapping signals from complex ion distributions. Electron-capture charge reduction (ECCR), in which a protein cation captures free electrons without apparent dissociation, can dealign overlapping signals by shifting the ions to lower charge states. The concomitant shift to higher m/z also facilitates the exploration of instrument upper m/z limits if large complexes are used. Here we perform native ECCR on the bacterial chaperonin GroEL and megadalton scale adeno-associated virus (AAV) capsid assemblies on the Q Exactive UHMR mass spectrometer. Charge reduction of AAV8 capsids by up to 90% pushes signals well above 100,000 m/z and enables charge state resolution and mean mass determination of these highly heterogeneous samples, even for capsids loaded with genetic cargo. With minor instrument modifications the UHMR can detect charge reduced ion signals beyond 200,000 m/z. This work demonstrates the utility of ECCR for deconvolving heterogeneous signals in native mass spectrometry and presents the highest m/z signals ever recorded on an Orbitrap instrument, opening up the use of Orbitrap native mass spectrometry for heavier analytes than ever before.

Protein complex heterogeneity and topology revealed by electron capture charge reduction and surface induced dissociation

“…Our current results (https://www.biorxiv.org/content/10.1101/2024.03.07.583498v1) 74,75 are in strong agreement with the related work of Sobott and coworkers. 51,52 We use the acronym ECCR to describe this gas phase charge transfer 53,74,75 and agree with Sobott and coworkers who adopted the phrase and suggest 52 that it should be used when investigators use electron capture to induce charge reduction (in contrast to using the phrase electron capture dissociation, which describes experiments where fragments resulting from covalent cleavage are the intended products). Furthermore, we demonstrated that gas-phase charge reduction produces charge states that give native-like surface induced dissociation fragments for C-reactive protein and GroEL.…”

“…These results were reproduced and expanded upon in the recent ECCR results for GroEL shown by Sobott, Makarov, Fort, and co-workers. 51,52 Figure S3 demonstrates long-term stability and high reproducibility of ECCR for CRP, where it was observed the standard deviation in the average charge state after ECCR (± 1.9) is slightly higher than the standard deviation in the average charge state before ECCR (± 0.9), over 7 months of data acquisition.…”

“…Sobott and coworkers have demonstrated the utility of electron-based charge reduction to resolve heterogeneous oligomers of αβ-crystallin. 49 Ujma and co-workers, and more recently Sobott and co-workers, have shown its utility to resolve AAV capsids, 50–52 while Wysocki and coworkers have shown its ability to resolve fragments of AAV capsids. 53…”