- From Protein Complexes to Subunit Backbone Fragments: A Multistage Approach to Native Mass Spectrometry by Mikhail E. Belov,†,§ Eugen Damoc,† Eduard Denisov,† Philip D. Compton,‡ Stevan Horning,†Alexander A. Makarov,*,† and Neil L. Kelleher*,‡
Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS3, from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highlycharged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.
Spectroglyph, LLC received a grant, ref 1R43GM105152-01A1, from the National Institutes of Health to develop a Novel Hybrid Bioanalytical System.