Triple-Stage Mass Spectrometry Unravels the Heterogeneity of an Endogenous Protein Complex
Open accessAbstract
Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.
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In complex-down, the ejected ion (monomer or ligand) is subsequently re-isolated and fragmented in a pseudo-MS3 or MS4 workflow, providing in-depth information on phosphorylation sites, metal ion binding, or sequencing of peptide ligands.
“The ejected ion – monomer or ligand – is subsequently (re-)isolated and fragmented in a (pseudo-)MS3 or MS4 workflow, providing in-depth information on e.g., phosphorylation sites,104 metal ion binding,36 or sequencing of peptide ligands (see Fig. 3).105”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [104]
The FBP1 complex is deactivated under glucose growth conditions and the cells readily switch from performing gluconeogenesis to glycolysis.
“indicating that the complex is deactivated under these conditions and the cells readily switch from performing gluconeogenesis to glycolysis.”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [104]
Complex-down analysis of FBP1 monomers indicated that phosphorylation on Ser12 or Thr13 was highly expressed in cells grown on glucose media.
“Complex-down analysis of the monomers indicated that phosphorylation on Ser12 or Thr13 was highly expressed in cells that were grown on glucose media.”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [104]
The level of phosphorylation of the fructose-1,6-bisphosphatase 1 (FBP1) complex differed depending on whether yeast cells were grown on carbon-starved media, glucose media, or were heat-shocked.
“It was found that the level of phosphorylation of the fructose-1,6-bisphosphatase 1 (FBP1) complex differed depending on whether the cells were grown on carbon-starved media, glucose media, or were heat-shocked.”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [104]
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Self-citation
- Authors
- Ben-Nissan, G., Belov, M. E., Morgenstern, D., Levin, Y., Dym, O., Arkind, G., Lipson, C., Makarov, A. A., & Sharon, M.
- Affiliations
- Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel
- Thermo Fisher Scientific (Germany) (DE)
- Weizmann Institute of Science (IL)
- Corresponding author
- Sharon, Michal
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2017-04-06
- Volume
- 89
- Issue
- 8
- Pages
- 4708-4715
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC5702261
- OpenAlex
- W2603495890
- Cited by
- 54
- References
- 39
- Citations by year
- 2026: 2 · 2025: 2 · 2024: 3 · 2023: 5 · 2022: 2 · 2021: 8 · 2020: 10 · 2019: 10 · 2018: 3 · 2017: 8 · 2015: 1
- FWCI
- 4.18
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- H2020 European Research Council (636752)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- native mass spectrometry, multistage mass spectrometry, Orbitrap mass spectrometer, linear ion trap, gluconeogenesis, protein phosphorylation, stoichiometry, protein modification, yeast, protein assembly
- MeSH terms
- Fructose-Bisphosphatase, Phosphorylation, Saccharomyces cerevisiae, Mass Spectrometry, Temperature, Protein Subunits, Saccharomyces cerevisiae Proteins
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