Abstract
Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.
Highlights
Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes
We devised this method to circumvent two confounding issues: (1) The anion-exchange column chromatography approach previously introduced for the separation of glycerophosphoinositol phosphates cannot be adapted for ESI-MS/MS due to the requirement for a non-volatile phosphate buffer
The PRMC-MS approach described here enables the comprehensive analysis of phosphoinositide acyl variants in various types of biological samples, including cultured cells, CM, exosomes, isolated tissues, and blood
Summary
Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Combinatorial phosphorylation of residues in the PI head group gives rise to seven other phosphoinositide classes, namely PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3. These lipids spatiotemporally control the activities of many proteins possessing phosphoinositide-binding motifs. Genetic alterations in phosphoinositide-metabolizing enzymes, including kinases, phosphatases, lipases, and acyltransferases, have been implicated in the pathogenesis of various diseases[4,5]
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