ABSTRACT
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.
