Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry

Abstract

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (≥10(5)) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

Figure 1

Analysis of phosphoinositides in control and fMLP-stimulated human neutrophils. (a-d) Neutral loss scans of a derivatised phosphoinositide extract, from un-stimulated control (a, c) or fMLP-stimulated (b, d) neutrophils, ISD = internal standard. The two most abundant species of endogenous PtdIns(3,4,5)P₃ and PtdInsP₂ are labeled with full masses and corresponding fatty acid species of DAG unit. (e) Overlay of m/z chromatograms for parent ions with masses similar to derivatised C18:0/C20:4-Ptdins(3,4,5)P₃ from extracts from 1 ×10⁵ human neutrophils, highlighting elution at 10.75 min (ions which increase with fMLP-stimulation in a wortmannin-sensitive manner). (f) Overlay of MRM chromatograms (m/z 1,225 → m/z 627 + 598) of samples from e. Data in a-d were collected using a Quattro Ultima mass spectrometer and data in e-f were collected using a QTRAP4000 mass spectrometer. Each are representative traces from several independent experiments. A full list of relevant structures and associated masses are shown in Supplementary Table 1; sn-1/sn-2 assignment is based on biological precedent and further fragmentation of DAG species.

Figure 2

Validation of the robustness, signal-to-noise and linearity of the assay. a) C17:0/C16:0-PtdIns(3,4,5)P₃ internal standard (ISD) spiked into un-stimulated human neutrophil extract; (b) C18:0/C20:4-PtdIns(3,4,5)P₃ standard spiked into un-stimulated human neutrophil extract; (c) 0.25 ng C18:0/C20:4-PtdIns(3,4,5)P₃ standard spiked into un-stimulated neutrophil extract; (d) linear response to increasing amounts of C18:0/C20:4-PtdIns(3,4,5)P₃ added to extracts of un-stimulated neutrophils (2.25 × 10⁶); (e) linear relationship between cell number (fMLP-stimulated neutrophils) and estimated endogenous C18:0/C20:4-PtdIns(3,4,5)P₃ by using the ISD to correct for recovery. The term ‘response ratio’ in (b,d,e) is the integrated ion current response to the defined phosphoinositide divided by that to the ISD. (f) MRM chromatograms for 1μg each of synthetic C18:0/C20:4-PtdIns(4,5)P₂ (i), synthetic C18:0/C20:4-PtdIns(3,4,5)P₃ (ii) and ISD (C17:0/C16:0-PtdIns(3,4,5)P₃) (iii) in water, demonstrating equal recovery/detection.

Figure 3

fMLP-stimulated changes in C18:0/C20:4 and C18:0/C18:1 PtdInsP₂ and PtdIns(3,4,5)P₃ species in human neutrophils. (a-b) The levels of the C18:0/C20:4 and C18:0/C18:1 molecular species of PtdInsP₂ (a) and PtdIns(3,4,5)P₃ (b) in human neutrophils were determined at a range of times following addition of fMLP. The data are expressed as either response ratios (as defined in fig. 2) or, through use of the calibration curve presented in Fig. 2d, and protein assays, pmol mg⁻¹ protein (Y-axis, right) and are means ± SEM (n= 4). (c) The ratio of the quantity of each molecular species of PtdIns(3,4,5)P₃ divided by that of their respective PtdInsP₂ species.

Figure 4

Identification and quantification of the molecular species of PtdIns(3,4,5)P₃ in wild-type and PTEN^−/− MCF10a cells. (a-b) Neutral loss scans of the common families of molecular species of PtdInsP₂ (a) and PtdIns(3,4,5)P₃ (b) in EGF-stimulated, wild-type MCF10a cells, Further fragmentation and analysis of the relevant daughter ions indicated they possessed the fatty acids indicated in c,d. Data was collected using a QTRAP 4000 mass spectrometer. (c-d) The levels of these species of PtdInsP₂ (c) and PtdIns(3,4,5)P₃ (d) in the indicated cell lines and conditions are presented as mean response ratios normalized for cell input via the recovered C18:0/C18:1-PtdSer (means ± SEM, n= 3). (e) The ratio of the quantity of each molecular species of PtdIns(3,4,5)P₃ divided by that of their respective PtdInsP₂ species. Full details of molecular species, and masses of respective parent ions detected in MCF10a cells can be found in Supplementary Table 1.

Figure 5

Detection and quantification of insulin-stimulated PtdIns(3,4,5)P₃ responses in mouse liver and human adipose tissue. (a) Neutral loss scan, collected using a QTRAP 4000 mass spectrometer, of PtdIns(3,4,5)P₃ species in wild-type (WT), insulin-stimulated mouse liver. (b) Levels of C18:0/C20:4-PtdIns(3,4,5)P₃ in the livers of WT or Gnasxl^m+/p− mice, after injection of insulin or saline (means ± SEM, n= 4). (c) Phosphorylation status of S473 in PKB for parallel samples to those analyzed in b; data normalized for input material via immuno-blotting for β-COP (See Supplementary Fig. 10 for blot). (d) Neutral loss scan of PtdIns(3,4,5)P₃ species in human adipose tissue following oral ingestion of glucose. (e) Levels of C18:0/C20:4-PtdIns(3,4,5)P₃ in healthy human adipose tissue following overnight starvation either before (Fasting) or 90 mins after (Post-Glucose) oral ingestion of glucose, for three individuals (means ± SEM, technical replicates n= 4). (f) Phosphorylation status of S473 in PKB for parallel samples to those analyzed in e; data normalized for input material via immuno-blotting for actin (See Supplementary Fig. 10 for blot).