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Top-down characterization of endogenous protein complexes with native proteomics
Open accessAbstract
Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.
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In the citing paper
“Through this lens, the number and variety of proteoforms expressed in biological systems appears to be well below the theoretical combinatorial possibilities 31 , with several examples providing a glimpse into this open question (Table 1).”
From How many human proteoforms are there? (2018) · cites this paper as [31]
Experimental — accuracy not yet measuredIn the citing paper
“Using Native GELFrEE and IEX, Skinner et al. identified 125 intact endogenous complexes from mouse hearts and human cancer cell lines [89].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [89]
In the citing paper
“In recent years, important progress in native purification methods such as native gel-eluted liquid fraction entrapment electrophoresis (GeLFrEE) separation,36 native gel electrophoresis,37 ion exchange chromatography (IEX),38 hydrophobic interaction chromatography (HIC),39,40 and online buffer exchange, has made these methods more applicable for complex mixtures and for characterisation of endo”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [36]
In the citing paper
“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 [36]
In the citing paper
“Recently, high-throughput native separation techniques including GELFrEE,36 HIC,39,40 SEC,112 capillary zone electrophoresis (CZE),112 and IEX38 have efficiently aided in the analysis of heterogeneous protein mixtures.”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [36]
In the citing paper
“indicating that phosphorylation at serine 20 and N-terminal acetylation act to inhibit the dimerisation of the complex.36”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [36]
In the citing paper
“The phosphorylated and acetylated proteoforms did not dimerise with themselves or each other, indicating that phosphorylation at serine 20 and N-terminal acetylation act to inhibit the dimerisation of the complex.36”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [36]
In the citing paper
“Compton and co-workers demonstrated how complex-down analysis of proteins with regulatory post-translational modifications leads to more accurate structural and functional characterisation of those complexes.36”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [36]
In the citing paper
“Sequence analysis of the triosephosphate isomerase complex indicated the presence of a proteoform that was not modified, a proteoform that was phosphorylated at serine 20, and a proteoform that was N-terminally acetylated.”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [36]
In the citing paper
“Interestingly, GAPDH has been observed not to have many proteoforms by top-down analysis.(21)”
From Putting humpty dumpty back together again: what does protein quantification mean in bottom-up proteomics? (2022) · cites this paper as [21]
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Metadata
- Authors
- Skinner, O. S., Haverland, N. A., Fornelli, L., Melani, R. D., Do Vale, L. H. F., Seckler, H. S., Doubleday, P. F., Schachner, L. F., Srzentić, K., Kelleher, N. L., & Compton, P. D.
- Affiliations
- Northwestern University (US)
- Corresponding author
- Kelleher, Neil L
- Compton, Philip D
- ORCID
- Venue
- Nature Chemical Biology
- Published
- 2017-11-13
- Volume
- 14
- Issue
- 1
- Pages
- 36-41
- ISSN
- 1552-4450 (ISSN-L)
- 1552-4469
- PMC
- PMC5726920
- OpenAlex
- W2768727863
- Cited by
- 155
- References
- 53
- Citations by year
- 2026: 12 · 2025: 16 · 2024: 15 · 2023: 16 · 2022: 16 · 2021: 25 · 2020: 20 · 2019: 22 · 2018: 12
- FWCI
- 7.42
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Chemical Biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (P41GM108569, T32GM105538)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Keywords
- protein complexes, protein-protein interaction, post-translational modification, covalent modification, ligand binding, manganese superoxide dismutase, metal cofactors, proteoforms, mouse heart, cancer cell lines
- MeSH terms
- Animals, Humans, Protein Conformation, Protein Processing, Post-Translational, Protein Subunits, Proteomics, Cell Line, Tumor, Multiprotein Complexes, Mice, Tandem Mass Spectrometry, Protein Multimerization
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