Functionally Integrated Top-Down Proteomics for Standardized Assessment of Human Induced Pluripotent Stem Cell-Derived Engineered Cardiac Tissues.

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.

Top-Down Proteomics of Endogenous Membrane Proteins Enabled by Cloud Point Enrichment and Multidimensional Liquid Chromatography-Mass Spectrometry.

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.