A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages.
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.
Pasquali
Christian
C
Merck Serono International S.A., 9 Ch. Des Mines, 1202 Geneva, Switzerland.
Bertschy-Meier
Dominique
D
Chabert
Christian
C
Curchod
Marie-Laure
ML
Arod
Christian
C
Booth
Randy
R
Mechtler
Karl
K
Vilbois
Francis
F
Xenarios
Ioannis
I
Ferguson
Colin G
CG
Prestwich
Glenn D
GD
Camps
Montserrat
M
Rommel
Christian
C
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008055:Lipids / Q000737:chemistry; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction
D047908:Intracellular Signaling Peptides and Proteins; D008055:Lipids; D010716:Phosphatidylinositols; D019869:Phosphatidylinositol 3-Kinases