Protein Complex Heterogeneity and Topology Revealed by Electron Capture Charge Reduction and Surface Induced Dissociation

Jared B Shaw; Sophie R Harvey; Chen Du; Zhixin Xu; Regina M Edgington; Eduardo Olmedillas; Erica Ollmann Saphire; Vicki H Wysocki

doi:10.1021/acscentsci.4c00461

Protein Complex Heterogeneity and Topology Revealed by Electron Capture Charge Reduction and Surface Induced Dissociation

ACS Cent Sci. 2024 Jul 26;10(8):1537-1547. doi: 10.1021/acscentsci.4c00461. eCollection 2024 Aug 28.

Authors

Jared B Shaw¹, Sophie R Harvey², Chen Du^{2

3}, Zhixin Xu^{2

3}, Regina M Edgington³, Eduardo Olmedillas⁴, Erica Ollmann Saphire^{4

5}, Vicki H Wysocki^{2

3}

Affiliations

¹ Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588, United States.
² Native Mass Spectrometry Guided Structural Biology Center, Ohio State University, Columbus, Ohio 43210, United States.
³ Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210, United States.
⁴ Center for Vaccine Innovation, La Jolla Institute for Immunology, La Jolla, California 92037, United States.
⁵ Department of Medicine, University of California San Diego, La Jolla, California 92037, United States.

Abstract

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.