Direct LC-MS/MS Analysis of Extra- and Intracellular Glycerophosphoinositol in Model Cancer Cell Lines

Abstract

Glycerophosphoinositols (GPIs) are water-soluble bioactive phospholipid derivatives of increasing interest as intracellular and paracrine mediators of eukaryotic cell functions. The most representative compound of the family is glycerophosphoinositol (GroPIns), an ubiquitous component of mammalian cells that participates in cell proliferation, cell survival and cell response to stimuli. Levels and activity of this compound vary among cell types and deciphering these functions requires accurate measurements in in vitro and in vivo models. The conventional approaches for the analysis of GroPIns pose several issues in terms of sensitivity and product resolution, especially when the product is in the extracellular milieu. Here we present an UPLC-MS study for the quantitative analysis of this lipid derivative in cells and, for the first time, culture supernatants. The method is based on a solid-phase extraction that allows for fast desalting and analyte concentration. The robustness of the procedure was tested on the simultaneous measurements of intra- and extracellular levels of GroPIns in a number of human cell lines where it has been shown that the non-transformed cells are characterized by high extracellular level of GroPIns, whereas the tumor cells tended to have higher intracellular levels.

Keywords: autocrine and paracrine mechanism; cancer; inflammation; lipid metabolism; mass spectrometry; second messenger; solid phase extraction.

Figure 1

MS Analysis of GroPIns. (A) Total ion chromatographic profile of cell extracts (MDA-MB-231); (B) Selected ion chromatogram (HRESI⁻ m/z 333.0594) of GroPIns; (C) MS/MS spectrum of GroPIns: HRESI⁻ m/z 333.0593 [M]⁻, 241.0108 [M-glycerol]⁻, 152.9951 [M-inositol]⁻, 96.9683 [H₂${\text{PO}}_4^{-}$]⁻ and 78.9572 [PO₃]⁻; (D) calibration curve in milli-Q water; (E) calibration curve in 2% formic acid.

Figure 2

GroPIns levels in non-tumorigenic and tumor-derived cell lines. GroPIns levels on (A) human prostate immortalized PNT2 and tumor-derived PC3 prostate cell lines, and (B) human mammary epithelial MCF10A and tumor-derived MDA-MB-231 cell lines. Quantitative data were obtained by external calibration method and normalized by 1 × 10⁵ cells. Total levels correspond to the mathematical sum of intracellular and extracellular values. Measurements were made in triplicate (n = 3). ****p ≤ 0.0001; ***p ≤ 0.001.

Figure 3

Effect of cPLA₂α inhibition on GroPIns intracellular and extracellular levels. A375MM cells were either untreated or exposed for 16 h to 0.5 μM cPLA₂α inhibitor. Quantitative GroPIns data were obtained by external calibration method and normalized per 1 × 10⁵ cells. Total levels correspond to the mathematical sum of intracellular and extracellular values. Measurements were made in triplicate (n = 3). ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.