Middle‐down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post‐translational modifications in histones
Abstract
We present an integrated middle‐down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5–7 kDa). We combined an RP trap column with subsequent weak cation exchange–hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N‐terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 ( Suz12 −/− ) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven‐fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12 − / − cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.
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Recent estimates from middle-down studies suggest that ~1,000 proteoforms exist for each of the H3 genes.
“Recent estimates from middle-down studies suggest that ~1,000 proteoforms exist for each of the H3 genes 50,92 .”
From How many human proteoforms are there? (2018) · cites this paper as [92]
Cites
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How many human proteoforms are there?
Aebersold, R., Agar, J. N., Amster, I. J., Baker, M. S., Bertozzi, C. R., Boja, E. S., Costello, C. E., Cravatt, B. F., Fenselau, C., Garcia, B. A., et al. (2018) · Nature Chemical Biology · doi:10.1038/nchembio.2576 · Cited by 907 · see citing works
Self-citationEZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas
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Self-citationToward an Optimized Workflow for Middle-Down Proteomics
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Tvardovskiy, A., Schwämmle, V., Kempf, S. J., Rogowska-Wrzesinska, A., & Jensen, O. N. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx696 · Cited by 132 · see citing works
Self-citationSidoli, S., Lin, S., Karch, K. R., & Garcia, B. A. (2015) · Analytical Chemistry · doi:10.1021/acs.analchem.5b00072 · Cited by 67 · see citing works
Self-citationAnderson, L. C., Karch, K. R., Ugrin, S. A., Coradin, M., English, A. M., Sidoli, S., Shabanowitz, J., Garcia, B. A., & Hunt, D. F. (2016) · Molecular & Cellular Proteomics · doi:10.1074/mcp.o115.053843 · Cited by 60 · see citing works
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- Authors
- Sidoli, S., Schwämmle, V., Ruminowicz, C., Hansen, T. A., Wu, X., Helin, K., & Jensen, O. N.
- Affiliations
- University of Southern Denmark (DK)
- University of Copenhagen (DK)
- Corresponding author
- Jensen, Ole N.
- Venue
- PROTEOMICS
- Publisher
- Wiley
- Published
- 2014-08-28
- Volume
- 14
- Issue
- 19
- Pages
- 2200-2211
- ISSN
- 1615-9853 (ISSN-L)
- 1615-9861
- OpenAlex
- W1987863569
- Cited by
- 87
- References
- 6
- Citations by year
- 2026: 2 · 2025: 5 · 2024: 4 · 2023: 3 · 2022: 3 · 2021: 4 · 2020: 4 · 2019: 7 · 2018: 11 · 2017: 14 · 2016: 12 · 2015: 18
- FWCI
- 6.29
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- PROTEOMICS (publishedVersion)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (submittedVersion)
- University of Southern Denmark Research Portal (University of Southern Denmark) (submittedVersion)
- Funders
- Danish National Research Foundation (DNRF82)
- VILLUM Center for Bioanalytical Sciences
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- histone modifications, histone N-terminal tails, middle-down proteomics, electron transfer dissociation, histone H3, histone H4, histone H2A, H3K27 methylation, mouse embryonic stem cells
- MeSH terms
- Animals, Cell Line, Chromatography, High Pressure Liquid, Histones, Methylation, Protein Processing, Post-Translational, Computational Biology, Spectrometry, Mass, Electrospray Ionization, Mice, Embryonic Stem Cells, Tandem Mass Spectrometry
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