Mass spectrometry: From plasma proteins to mitochondrial membranes

Abstract

In this Inaugural Article, I trace some key steps that have enabled the development of mass spectrometry for the study of intact protein complexes from a variety of cellular environments. Beginning with the preservation of the first soluble complexes from plasma, I describe our early experiments that capitalize on the heterogeneity of subunit composition during assembly and exchange reactions. During these investigations, we observed many assemblies and intermediates with different subunit stoichiometries, and were keen to ascertain whether or not their overall topology was preserved in the mass spectrometer. Adapting ion mobility and soft-landing methodologies, we showed how ring-shaped complexes could survive the phase transition. The next logical progression from soluble complexes was to membrane protein assemblies but this was not straightforward. We encountered many pitfalls along the way, largely due to the use of detergent micelles to protect and stabilize complexes. Further obstacles presented when we attempted to distinguish lipids that copurify from those that are important for function. Developing new experimental protocols, we have subsequently defined lipids that change protein conformation, mediate oligomeric states, and facilitate downstream coupling of G protein-coupled receptors. Very recently, using a radical method—ejecting protein complexes directly from native membranes into mass spectrometers—we provided insights into associations within membranes and mitochondria. Together, these developments suggest the beginnings of mass spectrometry meeting with cell biology.

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…For nearly thirty years, ongoing efforts have endeavoured to use MS to understand noncovalent protein complexes by transferring them into the gas phase without loss of higher-order structure. 5,18 At its most basic level, native MS can return information on the makeup of protein complexes by providing molecular weights more accurately than conventional biophysical methods (e.g., size-exclusion chromatography (SEC) or analytical ultracentrifugation) especially for heterogeneous samples. In relatively simple cases, this can suffice to indicate the number of monomers in a complex and determine differences in complex makeup.…”

Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass-spectrometry

“…Over the last decades bimolecular mass spectrometry (MS), with its ability to analyze low amounts of samples with high speed and sensitivity, has evolved into a central pillar beneficial for integrative structural biology (de Souza and Picotti, 2020;Kaur et al, 2019;Lossl et al, 2016;Robinson, 2019). The structural MS toolbox contains multiple complementary approaches.…”

Surface plasmon resonance, Orbitrap mass spectrometry and Raman advancements: exciting new techniques in drug discovery

“…Orbitrap has been successfully used for both the approaches and facilitated the identification of challenging protein isoforms and post translational modifications (PTMs) indicating the high mass accuracy, resolving power, and performance [9]. Another major leap in the use of Orbitrap is native mass spectrometry for the discovery of noncovalent protein complexes in their native form [10]. Of the several advancements made in Orbitrap mass spectrometers since its invention, addition of alternative fragmentation techniques such as electron transfer dissociation has improved the possibilities of Orbitrap [11].…”