Chemical Proteomic Profiling of Lysophosphatidic Acid-Binding Proteins

Lysophosphatidic acid (LPA) is an endogenous cell signaling
molecule,
and dysregulation of LPA signaling pathways is accompanied by several
types of cancer. Herein, we developed a chemical proteomic method
for the proteome-wide identification of LPA-binding proteins. The
method involves the synthesis of a desthiobiotin-conjugated LPA acyl
phosphate probe for the covalent labeling, enrichment, and subsequent
LC-MS/MS identification of LPA-binding proteins at the proteome-wide
level. By conducting labeling reactions at two different probe concentrations
(10 and 100 μM) in conjunction with an SILAC (stable isotope
labeling by amino acids in cell culture)-based workflow, we characterized
the LPA-binding capabilities of these proteins at the entire proteome
scale, which led to the identification of 86 candidate LPA-binding
proteins in HEK293T cells. Moreover, we validated that two of these
proteins, annexin A5 and phosphoglycerate kinase 1, can bind directly
with LPA. Together, we developed a novel LPA probe for the identification
and characterizations of LPA-binding proteins from the entire human
proteome. The method should be adaptable for the identification of
other lipid-binding proteins.