Article: article from journal or magazin.
A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages.
Molecular and Cellular Proteomics
Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.
Animals, Cells, Cultured, Chromatography, Affinity, Intracellular Signaling Peptides and Proteins/chemistry, Intracellular Signaling Peptides and Proteins/isolation & purification, Lipids/chemistry, Macrophages/enzymology, Mice, Phosphatidylinositol 3-Kinases/metabolism, Phosphatidylinositols/chemistry, Proteomics/methods, Signal Transduction
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