High Sensitivity Top-down Proteomics Captures Single Muscle Cell Heterogeneity in Large Proteoforms

Abstract

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 reproducibly detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high mass accuracy to enable the classification of individual fiber types. This study represents the first single-cell top-down proteomics analysis that captures 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.

Recent methodological advances towards single-cell proteomics

“…In addition, the MS techniques utilized in previous decades were relatively weak in terms of the quantitative performance; peptides with different sequences have different efficiencies of ionization, for which intensities in a mass spectrum cannot accurately reflect their abundances. 102 ) If these limitations are solved by technical advances, mass imaging 103 – 105 ) and top-down MS 8 , 106 ) would be powerful tools to conduct protein identification and study additional biologically important information such as subcellular localization and various proteoforms, respectively.…”

Single‐cell proteomics by mass spectrometry: Advances and implications in cancer research

“…Current technical challenges in top-down proteomics, even in bulk proteomics, is the decay in signal-to-noise ratio with increasing molecular weight, which require more sophisticated MS with high resolving power, m/z accuracy, and new ionisation technique. As a growing field in MS, Melby et al introduced a top-down proteomics platform for SCP where they tested it on muscle fibres [121]. Such an attempt has enabled the discovery of proteoform heterogeneity between functionally distinct muscle fibres, which would have otherwise been masked by conventional bottom-up proteomics.…”

“…As a growing field in MS, Melby et al. introduced a top‐down proteomics platform for SCP where they tested it on muscle fibres [121]. Such an attempt has enabled the discovery of proteoform heterogeneity between functionally distinct muscle fibres, which would have otherwise been masked by conventional bottom‐up proteomics.…”

Structure and dynamics of endogenous protein complexes in human heart tissue captured by native nanoproteomics

“…These present tremendous challenges to studying their structure and dynamics using traditional structural biology techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM) 2 . Native top-down mass spectrometry (nTDMS), a technique combining native MS 3 – 5 and top-down proteomics 6 – 11 , has emerged as a powerful structural biology tool for characterization of protein complexes 12 – 17 . In nTDMS, intact proteins are introduced into the mass spectrometer under non-denaturing conditions, preserving the noncovalent interactions between protein subunits and their associated ligands as well as PTMs.…”