ABSTRACT
Three-dimensional (3D) human induced pluripotent stem cell-derived engineered cardiac tissues (hiPSC-ECTs) have emerged as a promising alternative to two-dimensional hiPSC-cardiomyocyte monolayer systems because hiPSC-ECTs are a closer representation of endogenous cardiac tissues and more faithfully reflect the relevant cardiac pathophysiology. The ability to perform functional and molecular assessments using the same hiPSC-ECT construct would allow for more reliable correlation between observed functional performance and underlying molecular events, and thus is critically needed. Herein, for the first time, we have established an integrated method that permits sequential assessment of functional properties and top-down proteomics from the same single hiPSC-ECT construct. We quantitatively determined the differences in isometric twitch force and the sarcomeric proteoforms between two groups of hiPSC-ECTs that differed in the duration of time of 3D-ECT culture. Importantly, by using this integrated method we discovered a new and strong correlation between the measured contractile parameters and the phosphorylation levels of alpha-tropomyosin between the two groups of hiPSC-ECTs. The integration of functional assessments together with molecular characterization by top-down proteomics in the same hiPSC-ECT construct enables a holistic analysis of hiPSC-ECTs to accelerate their applications in disease modeling, cardiotoxicity, and drug discovery. Data are available via ProteomeXchange with identifier PXD022814.
Subject(s)
Induced Pluripotent Stem Cells , Cardiotoxicity , Cell Differentiation , Humans , Myocytes, Cardiac , Proteomics , Tissue EngineeringABSTRACT
Although top-down proteomics has emerged as a powerful strategy to characterize proteins in biological systems, the analysis of endogenous membrane proteins remains challenging due to their low solubility, low abundance, and the complexity of the membrane subproteome. Here, we report a simple but effective enrichment and separation strategy for top-down proteomics of endogenous membrane proteins enabled by cloud point extraction and multidimensional liquid chromatography coupled to high-resolution mass spectrometry (MS). The cloud point extraction efficiently enriched membrane proteins using a single extraction, eliminating the need for time-consuming ultracentrifugation steps. Subsequently, size-exclusion chromatography (SEC) with an MS-compatible mobile phase (59% water, 40% isopropanol, 1% formic acid) was used to remove the residual surfactant and fractionate intact proteins (6-115 kDa). The fractions were separated further by reversed-phase liquid chromatography (RPLC) coupled with MS for protein characterization. This method was applied to human embryonic kidney cells and cardiac tissue lysates to enable the identification of 188 and 124 endogenous integral membrane proteins, respectively, some with as many as 19 transmembrane domains.
