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

作者：Tanxi Cai, Qingbo Shu, Junjie Hou, Peibin Liu, Lili Niu, Xiaojing Guo, Charles C. Liu, Fuquan Yang · 发表于：Analytical Chemistry · 年份：2014 · DOI：10.1021/ac503224j · 被引用次数：37 · 研究领域：Hemoglobinopathies and Related Disorders、Mass Spectrometry Techniques and Applications、Blood disorders and treatments

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. T...