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High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic m...

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Autores principales: Melby, Jake A., Brown, Kyle A., Gregorich, Zachery R., Roberts, David S., Chapman, Emily A., Ehlers, Lauren E., Gao, Zhan, Larson, Eli J., Jin, Yutong, Lopez, Justin R., Hartung, Jared, Zhu, Yanlong, McIlwain, Sean J., Wang, Daojing, Guo, Wei, Diffee, Gary M., Ge, Ying
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10175728/
https://www.ncbi.nlm.nih.gov/pubmed/37126723
http://dx.doi.org/10.1073/pnas.2222081120
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author Melby, Jake A.
Brown, Kyle A.
Gregorich, Zachery R.
Roberts, David S.
Chapman, Emily A.
Ehlers, Lauren E.
Gao, Zhan
Larson, Eli J.
Jin, Yutong
Lopez, Justin R.
Hartung, Jared
Zhu, Yanlong
McIlwain, Sean J.
Wang, Daojing
Guo, Wei
Diffee, Gary M.
Ge, Ying
author_facet Melby, Jake A.
Brown, Kyle A.
Gregorich, Zachery R.
Roberts, David S.
Chapman, Emily A.
Ehlers, Lauren E.
Gao, Zhan
Larson, Eli J.
Jin, Yutong
Lopez, Justin R.
Hartung, Jared
Zhu, Yanlong
McIlwain, Sean J.
Wang, Daojing
Guo, Wei
Diffee, Gary M.
Ge, Ying
author_sort Melby, Jake A.
collection PubMed
description Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top–down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single-cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top–down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems.
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spelling pubmed-101757282023-11-01 High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms Melby, Jake A. Brown, Kyle A. Gregorich, Zachery R. Roberts, David S. Chapman, Emily A. Ehlers, Lauren E. Gao, Zhan Larson, Eli J. Jin, Yutong Lopez, Justin R. Hartung, Jared Zhu, Yanlong McIlwain, Sean J. Wang, Daojing Guo, Wei Diffee, Gary M. Ge, Ying Proc Natl Acad Sci U S A Biological Sciences Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top–down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single-cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top–down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems. National Academy of Sciences 2023-05-01 2023-05-09 /pmc/articles/PMC10175728/ /pubmed/37126723 http://dx.doi.org/10.1073/pnas.2222081120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Melby, Jake A.
Brown, Kyle A.
Gregorich, Zachery R.
Roberts, David S.
Chapman, Emily A.
Ehlers, Lauren E.
Gao, Zhan
Larson, Eli J.
Jin, Yutong
Lopez, Justin R.
Hartung, Jared
Zhu, Yanlong
McIlwain, Sean J.
Wang, Daojing
Guo, Wei
Diffee, Gary M.
Ge, Ying
High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
title High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
title_full High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
title_fullStr High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
title_full_unstemmed High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
title_short High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
title_sort high sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10175728/
https://www.ncbi.nlm.nih.gov/pubmed/37126723
http://dx.doi.org/10.1073/pnas.2222081120
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