Enhancing Identifications of Lipid-embedded Proteins by Mass Spectrometry for Improved Mapping of Endothelial Plasma Membranes in Vivo*S⃞

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American Society for Biochemistry and Molecular Biology

RESUMO

Lipid membranes structurally define the outer surface and internal organelles of cells. The multitude of proteins embedded in lipid bilayers are clearly functionally important, yet they remain poorly defined. Even today, integral membrane proteins represent a special challenge for current large scale shotgun proteomics methods. Here we used endothelial cell plasma membranes isolated directly from lung tissue to test the effectiveness of four different mass spectrometry-based methods, each with multiple replicate measurements, to identify membrane proteins. In doing so, we substantially expanded this membranome to 1,833 proteins, including >500 lipid-embedded proteins. The best method combined SDS-PAGE prefractionation with trypsin digestion of gel slices to generate peptides for seamless and continuous two-dimensional LC/MS/MS analysis. This three-dimensional separation method outperformed current widely used two-dimensional methods by significantly enhancing protein identifications including single and multiple pass transmembrane proteins; >30% are lipid-embedded proteins. It also profoundly improved protein coverage, sensitivity, and dynamic range of detection and substantially reduced the amount of sample and the number of replicate mass spectrometry measurements required to achieve 95% analytical completeness. Such expansion in comprehensiveness requires a trade-off in heavy instrument time but bodes well for future advancements in truly defining the ever important membranome with its potential in network-based systems analysis and the discovery of disease biomarkers and therapeutic targets. This analytical strategy can be applied to other subcellular fractions and should extend the comprehensiveness of many future organellar proteomics pursuits.

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