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 pro-teoforms arising from genetic mutations, alternative splicing, and post-translational mod-ifications. 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 heter-ogeneity 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 (similar to 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.

Automated summary

Single-cell proteomics is a powerful method to study cellular heterogeneity and functional networks. However, it faces challenges in analyzing proteoforms from genetic mutations, alternative splicing, and post-translational modifications. In this study, a sensitive top-down proteomics method was developed to analyze proteoforms from single muscle fibers. The study identified heterogeneity in large proteoforms and a relationship between sarcomeric proteoforms and muscle fiber types.