Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy
Open accessAbstract
Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances “degronomics,” i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3.
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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 [105]
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Zhou, M., Lantz, C., Brown, K. A., Ge, Y., Paša-Tolić, L., Loo, J. A., & Lermyte, F. (2020) · Chemical Science · doi:10.1039/d0sc04392c · Cited by 146 · see citing works
- Authors
- Canzani, D., Rusnac, D.-V., Zheng, N., & Bush, M. F.
- Affiliations
- University of Washington (US)
- Howard Hughes Medical Institute, Department of Pharmacology, University of Washington, Seattle, Washington 98195, United States
- Howard Hughes Medical Institute (US)
- Corresponding author
- Bush, Matthew F.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2019-09-17
- Volume
- 91
- Issue
- 20
- Pages
- 12775-12783
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC6959985
- OpenAlex
- W2974257266
- Cited by
- 11
- References
- 52
- Citations by year
- 2026: 2 · 2024: 3 · 2023: 2 · 2022: 2 · 2020: 2
- FWCI
- 0.33
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Division of Chemistry (CHE-1807382)
- Howard Hughes Medical Institute
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- E3 ubiquitin ligase, substrate recognition, peptidome, protein degradation, native top-down mass spectrometry, native mass spectrometry, Escherichia coli, diglycine, substrate specificity
- MeSH terms
- Amino Acid Sequence, Antigens, Neoplasm, Chromatography, High Pressure Liquid, Escherichia coli, Glycylglycine, Humans, Peptides, Protein Binding, Mass Spectrometry
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