Mapping intact protein isoforms in discovery mode using top-down proteomics
Open accessAbstract
A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
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The count of experimentally determined proteoforms was about ten times higher than in earlier reports.
“The number of experimentally determined proteoforms exceeded that of previous reports by ~10-fold (16)”
From The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells (2022) · cites this paper as [16]
Top-down proteomics avoids inferring proteins from shotgun peptide data and provides cell- and proteoform-specific information.
“In this study, we used top-down proteomics (TDP) (16), which avoids the problem of inferring proteins using peptide data from shotgun proteomics analysis (17), to obtain cell- and proteoform-specific information (18) from the major cell types present in the blood and bone marrow.”
From The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells (2022) · cites this paper as [16]
The Kelleher group combined solution isoelectric focusing, GELFrEE, and RPLC-MS/MS to identify ~3,000 proteoforms from 1043 protein groups including proteins up to 105 kDa.
“In 2012, the Kelleher group combined solution isoelectric focusing, GELFrEE, and RPLC-MS/MS to identify ~3,000 proteoforms from 1043 protein groups including proteins up to 105 kDa [84].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [84]
Global top-down proteomics using the Orbitrap has enabled identification and quantification of thousands of proteoforms.
“Moreover, global top-down proteomics using the Orbitrap has enabled the identification and quantification of thousands of proteoforms [84,100].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [84]
Top-down proteomics has made recent progress on high-mass proteins and complex samples, but limitations remain in sequence coverage and in analyzing low-abundance species.
“Although great strides have recently been made in the topdown analysis of high-mass proteins (7) and complex proteomic samples (8), limitations remain to be addressed in the degree of sequence coverage and the ability to analyze low-abundance species.”
From Proteoforms as the next proteomics currency (2018) · cites this paper as [8]
GPF can contribute to top-down proteomics in a similar manner to sophisticated multidimensional liquid phase separations.
“demonstrating that GPF can contribute to top-down proteomics in a similar manner to sophisticated multidimensional liquid phase separations.49-51”
From Proton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms (2019) · cites this paper as [51]
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- Authors
- Tran, J. C., Zamdborg, L., Ahlf, D. R., Lee, J. E., Catherman, A. D., Durbin, K. R., Tipton, J. D., Vellaichamy, A., Kellie, J. F., Li, M., Wu, C., Sweet, S. M. M., Early, B. P., Siuti, N., LeDuc, R. D., Compton, P. D., Thomas, P. M., & Kelleher, N. L.
- Affiliations
- University of Illinois Urbana-Champaign (US)
- Northwestern University (US)
- Harvard University (US)
- University of Wisconsin–Madison (US)
- Corresponding author
- Kelleher, Neil L.
- ORCID
- Venue
- Nature
- Published
- 2011-10-30
- Volume
- 480
- Issue
- 7376
- Pages
- 254-258
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC3237778
- OpenAlex
- W2012671659
- Cited by
- 665
- References
- 47
- Citations by year
- 2026: 25 · 2025: 24 · 2024: 14 · 2023: 28 · 2022: 28 · 2021: 46 · 2020: 32 · 2019: 49 · 2018: 43 · 2017: 69 · 2016: 61 · 2015: 53 · 2014: 59 · 2013: 74 · 2012: 57
- FWCI
- 27.68
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Figshare (submittedVersion, cc-by)
- Funders
- David and Lucile Packard Foundation
- Camille and Henry Dreyfus Foundation
- National Institute on Drug Abuse (F30DA026672, P30DA018310)
- National Institute of General Medical Sciences (R01GM067193, GM 067193-08)
- 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 › Metabolomics and Mass Spectrometry Studies
- Keywords
- top-down proteomics, protein isoforms, post-translational modification, human proteome, cellular senescence, DNA damage, alternative splicing, proteolysis, tandem mass spectrometry, Swiss-Prot
- MeSH terms
- Cell Line, DNA Damage, HeLa Cells, Humans, Phenotype, Protein Processing, Post-Translational, Cellular Senescence, Alternative Splicing, Protein Isoforms, Proteome, HMGA1a Protein, HMGA1b Protein, Databases, Protein, Proteomics, Proteolysis
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