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 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.

Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms

“…The observed mass corresponds to the theoretical MW of myosin heavy chain (MHC), a prominent thick filament protein in the sarcomere. , Although precursor ions were selected for fragmentation, the high charge and resulting low S/N prevented the detection of resolved tandem MS fragments given the small amount of sample used here. Thus, we validated the raw m / z spectrum to ensure the charge state distribution is consistent with the MW of MHC, similar to what was done in a previous study . Furthermore, the detection of MHC in F1 is consistently observed in similar elution windows across six different biological replicates (Figure S5).…”

“…Thus, we validated the raw m/z spectrum to ensure the charge state distribution is consistent with the MW of MHC, similar to what was done in a previous study. 27 Furthermore, the detection of MHC in F1 is consistently observed in similar elution windows across six different biological replicates (Figure S5). In F2 (blue), we noticed an additional chromatographic profile alteration during the 28.1−28.7 min elution window (Figure 2E).…”

“…By collecting 1 min fractions directly in LC-MS vials, there was no additional sample transfer step needed prior to MS analysis. Importantly, the concentrations of each fraction (Figure S3) were sufficient for our high sensitivity RPLC-MS/MS setup using a capillary column and the Newomics MnESI source . Representative total ion chromatograms (TICs) corresponding to each fraction (F#) and the traditional one-dimensional (1D)-RPLC analysis displayed unique profiles, suggesting the presence of different proteins in each fraction and further validating the success of offline fractionation (Figure B).…”

“…Importantly, the concentrations of each fraction (Figure S3) were sufficient for our high sensitivity RPLC-MS/MS setup using a capillary column and the Newomics MnESI source. 27 Representative total ion chromatograms (TICs) corresponding to each fraction (F#) and the traditional one-dimensional (1D)-RPLC analysis displayed unique profiles, suggesting the presence of different proteins in each fraction and further validating the success of offline fractionation (Figure 2B). Additionally, the chromatographic profiles of each fraction were consistent across different biological replicates, demonstrating the reproducibility of both the offline fractionation and the online RPLC-MS analysis (Figure S4).…”

Single Cell Analysis of Proteoforms

“…Despite significant advances, the rate of single cell processing is commonly a few hundred per day, even from cell lines. , To increase throughput, avoid multistep processing and protein digestion, a more direct sampling approach could be advantageous for “top-down” proteoform detection in single cells . However, a scheme for MS-based intact proteoform SCP sets requirements for sensitivity and protein identification that have yet to be met. , …”