Capturing Cardiomyocyte Cell-to-Cell Heterogeneity via Shotgun Top-Down Proteomics

Individual cells have unique molecular compositions and thus, biological functions. Because tissues are made up of collections of different cell types, it is important to define “cell-to-cell” heterogeneity to understand the role heterogeneity plays in health and disease. Top-down proteomics provides information about proteoforms, yet its use has largely been limited to the analysis of bulk samples that contain thousands or millions of cells rather than individual cells. We describe a shotgun top-down proteomics strategy that employs a CellenONE device, reversed-phase liquid chromatography, and electron-transfer/higher-energy collision dissociation (EThcD) to evaluate cell-to-cell heterogeneity and to profile protein structural changes in single cardiomyocyte cells from a mouse heart. Individual cardiomyocytes were collected in a 384-well plate using a CellenONE device. Each well was filled with a solution composed of trifluoroethanol and dimethyl sulfoxide in 0.1% formic acid with protease and phosphatase inhibitor cocktails. The single cell lysates were analyzed using a nanocapillary LC coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher). Each single cell lysate was separated using a 60-minute linear gradient on a fused silica capillary packed in-house with C4 reversed-phase resin. The Orbitrap Fusion Lumos was operated in intact protein mode. EThcD was used as a protein ion dissociation mode. Raw files were processed using ProSight PD 4.2. From 95 individual cardiomyocyte cells, thousands of proteoforms were identified. Our analysis showed a high degree of proteoform heterogeneity among the individual cells. Most proteoforms were truncated, with numerous posttranslational modifications of cardiac relevance (e.g., crotonylation, phosphorylation, and succinylation). These results show that our approach is capable of profiling proteoforms in individual cells to uncover previously inaccessible molecular features of the cardiomyocyte proteome.