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Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics
Open accessPreprint — published as 10.1021/jasms.3c00072
Abstract
Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious requiring two hours of mass spectrometry time per single muscle fiber; 50 fibers would take approximately four days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 minutes total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 hours. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Sixty-five proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, muscle structure and regulation. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.
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Global quantification of mammalian gene expression control
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Metadata
- Authors
- Momenzadeh, A., Jiang, Y., Kreimer, S., Teigen, L. E., Zepeda, C. S., Haghani, A., Mastali, M., Song, Y., Hutton, A., Parker, S. J., Van Eyk, J. E., Sundberg, C. W., & Meyer, J. G.
- Affiliations
- Cedars-Sinai Medical Center (US)
- Marquette University (US)
- Corresponding author
- Sundberg, Christopher W.
- Meyer, Jesse G.
- ORCID
- Venue
- Journal of the American Society for Mass Spectrometry
- Publisher
- openRxiv
- Published
- 2023-02-23
- Volume
- 34
- Issue
- 9
- Pages
- 1858-1867
- ISSN
- 1044-0305 (ISSN-L)
- PMC
- PMC11135628
- OpenAlex
- W4384665127
- Cited by
- 28
- References
- 1
- Citations by year
- 2026: 9 · 2025: 9 · 2024: 7 · 2023: 3
- FWCI
- 2.58
- Type
- journal article
- Language
- en
- Open access
- green
- dx.doi.org
- Licence
- cc-by-nc-nd
- Locations
- Journal of the American Society for Mass Spectrometry (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- PubMed Central (submittedVersion, cc-by-nc-nd)
- e-Publications@Marquette (Marquette University) (submittedVersion)
- Funders
- National Institutes of Health
- National Institute on Aging (R01AG048262, R21AG074234)
- National Heart, Lung, and Blood Institute (R01HL111362)
- National Institute of General Medical Sciences (R35GM142502)
- 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 › Muscle Physiology and Disorders
- Keywords
- high-throughput proteomics, single-cell proteomics, fatty acid oxidation, oxidative phosphorylation, muscle contractility, human skeletal muscle
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