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Profiling and Relative Quantitation of Phosphoinositides by Multiple Precursor Ion Scanning Based on Phosphate Methylation and Isotopic Labeling

Cai, Tanxi, Shu, Qingbo, Hou, JunJie, Liu, Peibin, Niu, Lili, Guo, Xiaojing, Liu, Charles C., Yang, Fuquan
Analytical chemistry 2015 v.87 no.1 pp. 513-521
brain, cattle, cell growth, diacylglycerols, fatty acid composition, isotope labeling, mass spectrometry, metabolism, methanolysis, methylation, phosphates, prostatic neoplasms, rapid methods
Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), are key regulators of many fundamental biological processes, including cell growth, proliferation, and motility. Here, we present a novel method for rapid, sensitive, and simultaneous profiling of phosphatidylinositol trisphosphate (PtdInsP₃), phosphatidylinositol bisphosphate (PtdInsP₂), and phosphatidylinositol phosphate (PtdInsP) of different fatty acid compositions. This method is based on a technique called “charged diacylglycerol fragment ion-specific multiple precursor ion scanning” (DAG⁺-specific MPIS), coupled with prior phosphate methylation. Using DAG⁺-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30 PtdInsP₂, and 3 PtdInsP₃ molecular species from bovine brain extracts or prostatic cancer cell lines in an efficient and time-saving manner. Our analysis revealed a large range of fatty acyl compositions in phosphoinositides not obtained previously from mammalian samples. We also developed a method that involves isotopic labeling of endogenous phosphoinositides with deuterated diazomethane (CD₂N₂) for quantitation of phosphoinositides. CD₂N₂ was generated in situ through acid-catalyzed H/D exchange and methanolysis of trimethylsilyl diazomethane (TMS-diazomethane). Phosphoinositides, extracted from a PC3 prostatic cancer cell line, were labeled either with CH₂N₂ or CD₂N₂ and mixed in known proportions for DAG⁺-specific MPIS-based mass spectrometry (MS) analysis. The results indicate that isotopic labeling is capable of providing accurate quantitation of PtdInsP₃, PtdInsP₂, and PtdInsP with adequate linearity as well as high reproducibility with an average coefficient variation of 18.9%. More importantly, this new methods excluded the need for multiple phosphoinositide internal standards. DAG⁺-specific MPIS and isotopic labeling based MS analysis of phosphoinositides offers unique advantages over existing approaches and presents a powerful tool for research of phosphoinositide metabolism.