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

Abstract

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.

Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling

“…The quantitative analysis of PLs is usually achieved by the inclusion of multiple internal standards (ISDs) and run separately. An alternative strategy for quantifi cation of PLs would be to incorporate a given heavy nuclei (e.g., 2 H, 13 C, or 15 N) into endogenous PLs by metabolic or chemical labeling. Each population of PLs is reacted with a reagent that differs only in its isotopic composition, thereby creating "heavy" and "light" versions of derivatized PLs, which are distinguishable by MS.…”

“…Recently, Nie et al ( 12 ) also demonstrated isobaric mass stable isotope-labeled S,S ′ -dimethylthiobutanoylhydroxysucccinimide ester reagents for characterization and multiplexed quantifi cation of aminophospholipids in biological samples. The advantages of these strategies are that the use of MS2 for by guest, on May 11, 2018 www.jlr.org of individual species in the labeled and unlabeled lipid extracts, respectively, while I labeled and I unlabeled represent the height of the peak of the labeled and unlabeled PL species after 13 C correction, respectively. The results are presented as mean ± SD .…”

“…Due to the overlapping nature of the pseudomolecular ion peak of the species of interest with the M+2 isotope peak from another more unsaturated species that has a 2 Da lower mass (e.g., the 13 C isotope effect of PC 16:0/18:2 on PC 16:0/18:1), a 13 C correction was required for quantifi cation of the PLs. Moreover, due to a mass difference of 2 Da between the 1 H-and Dlabeled isotopic pairs for PL species, as well as their LPL counterparts, there would be an overlap between the unlabeled ion peak of the species of interest with the M+2 isotope peak and the labeled one (e.g., the 13 C isotope effect of unlabeled PE 16:0/18:0 on labeled PE 16:0/18:0).…”

“…Moreover, due to a mass difference of 2 Da between the 1 H-and Dlabeled isotopic pairs for PL species, as well as their LPL counterparts, there would be an overlap between the unlabeled ion peak of the species of interest with the M+2 isotope peak and the labeled one (e.g., the 13 C isotope effect of unlabeled PE 16:0/18:0 on labeled PE 16:0/18:0). Therefore, a two-step 13 C isotope correction was carried out for quantifi cation of the PLs.…”

“…However, they are limited to the relative quantitation of aminophospholipids. Previously, we have reported a strategy based on phosphate methylation and isotopic labeling for the relative quantitation of phosphoinositides from two different samples ( 13 ). This method involves isotopic labeling of endogenous phosphoinositides with deuterated diazomethane (CD 2 N 2 ) for quantitation of phosphoinositides.…”