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

ABSTRACT: Top-down proteomics has emerged as a powerful strategy to characterize proteins in biological systems. Nevertheless, the analysis of endogenous membrane proteins is challenging due to their low solubility, low abundance, and the complexity of the membrane subproteome. Here, we report a simple, effective enrichment and separation strategy for top-down proteomics of endogenous membrane proteins enabled by cloud-point enrichment 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 with an MS-compatible mobile phase was used to fractionate intact proteins (6-115 kDa). Fractions were separated further by reversed-phase liquid chromatography coupled with MS for protein characterization. We applied this method to human embryonic kidney cells and cardiac tissue, enabling the identification of 188 and 124 endogenous integral membrane proteins respectively, some with as many as 19 transmembrane domains.

INSTRUMENT(S): maXis

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Heart, Kidney

SUBMITTER: kyle brown  

LAB HEAD: Ying Ge

PROVIDER: PXD019368 | Pride | 2020-11-30

REPOSITORIES: Pride