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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.