Intact-Mass Analysis Facilitating the Identification of Large Human Heart Proteoforms
Open accessAbstract
Proteoforms, the primary effectors of biological processes, are the different forms of proteins that arise from molecular processing events such as alternative splicing and post-translational modifications. Heart diseases exhibit changes in proteoform levels, motivating the development of a deeper understanding of the heart proteoform landscape. Our recently developed two-dimensional top-down proteomics platform coupling serial size exclusion chromatography (sSEC) to reversed-phase chromatography (RPC) expanded coverage of the human heart proteome and allowed observation of high-molecular weight proteoforms. However, most of these observed proteoforms were not identified due to the difficulty in obtaining quality tandem mass spectrometry (MS2) fragmentation data for large proteoforms from complex biological mixtures on a chromatographic time scale. Herein, we sought to identify human heart proteoforms in this data set using an enhanced version of Proteoform Suite, which identifies proteoforms by intact mass alone. Specifically, we added a new feature to Proteoform Suite to determine candidate identifications for isotopically unresolved proteoforms larger than 50 kDa, enabling subsequent MS2 identification of important high-molecular weight human heart proteoforms such as lamin A (72 kDa) and trifunctional enzyme subunit α (79 kDa). With this new workflow for large proteoform identification, endogenous human cardiac myosin binding protein C (140 kDa) was identified for the first time. This study demonstrates the integration of our sSEC-RPC-MS proteomics platform with intact-mass analysis through Proteoform Suite to create a catalog of human heart proteoforms and facilitate the identification of large proteoforms in complex systems.
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A recently developed 2DLC method coupling serial size-exclusion chromatography with reversed-phase chromatography enabled the detection and identification of larger heart proteins, including cMyBP-C and MHC, but required much larger amounts of tissue than the HCM septal myectomy tissues obtained.
“Although we have recently developed a 2DLC method coupling serial size-exclusion chromatography with reversed-phase chromatography that enabled the detection and identification of larger heart proteins, including cMyBP-C and MHC (48, 49), it required much larger amount of tissues than the HCM septal myectomy tissues we obtained here.”
From Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics (2020) · cites this paper as [49]
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- Authors
- Schaffer, L. V., Tucholski, T., Shortreed, M. R., Ge, Y., & Smith, L. M.
- Affiliations
- University of Wisconsin–Madison (US)
- Corresponding author
- Ge, Ying
- Smith, Lloyd M.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2019-08-08
- Volume
- 91
- Issue
- 17
- Pages
- 10937-10942
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC6722015
- OpenAlex
- W2967062596
- Cited by
- 17
- References
- 33
- Citations by year
- 2026: 1 · 2024: 6 · 2023: 1 · 2022: 2 · 2021: 1 · 2020: 5 · 2019: 1
- FWCI
- 0.90
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Heart, Lung, and Blood Institute (R01HL096971, R01HL109810)
- Office of the Director (S10OD018475)
- National Institute of General Medical Sciences (R35GM126914, R01GM125085)
- 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
- proteoforms, high molecular weight proteins, myosin binding protein C, lamin A, protein identification, heart disease
- MeSH terms
- Myosin Binding Protein C, Amino Acid Sequence, Carrier Proteins, Chromatography, Gel, Humans, Myocardium, Protein Processing, Post-Translational, Software, Alternative Splicing, Proteome, Lamin Type A, Proteomics, Tandem Mass Spectrometry, Chromatography, Reverse-Phase, Mitochondrial Trifunctional Protein, alpha Subunit
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