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

Nature Methods volume 8, pages 267–272 (2011)Cite this article

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Abstract

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 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)P3 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)P3 and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP2 are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P3 in unstimulated mouse and human cells (≥105) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

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Figure 1: Analysis of phosphoinositides in control and fMLP-stimulated human neutrophils.
Figure 2: Validation of the robustness, signal-to-noise ratio and linearity of the assay.
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.
Figure 4: Identification and quantification of the molecular species of PtdIns(3,4,5)P3 in wild-type and PTEN−/− MCF10a cells.
Figure 5: Detection and quantification of insulin-stimulated PtdIns(3,4,5)P3 responses in mouse liver and human adipose tissue.

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Acknowledgements

We thank G. Kelsey (Babraham Institute) for providing mice, E. Ivanova for technical assistance and A. Segonds-Pichon for statistical analysis. V.J. acknowledges the support of the community under a Marie Curie Intra-European Fellowship for Career Development. This work was supported by the Biotechnology and Biological Sciences Research Council and European Union FP7 LipidomicNet (#202272).

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Author notes

  1. Jonathan Clark, Karen E Anderson, Len R Stephens and Phillip T Hawkins: These authors contributed equally to this work.

Authors and Affiliations

  1. Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, UK

    Jonathan Clark, Karen E Anderson, Veronique Juvin, Trevor S Smith, Michael J O Wakelam, Len R Stephens & Phillip T Hawkins

  2. Babraham Bioscience Technologies Ltd., Babraham Research Campus, Babraham, Cambridge, UK

    Jonathan Clark

  3. Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK

    Fredrik Karpe

  4. National Institute for Health Research, Oxford Biomedical Research Centre, Oxford Radcliffe Hospitals Trust, Churchill Hospital, UK

    Fredrik Karpe

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  1. Jonathan Clark
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Contributions

J.C. and K.E.A. designed and performed experiments, developed methods, analyzed data and contributed to writing the manuscript; V.J. designed and performed PKB experiments; T.S.S. performed experiments; F.K. designed experiments (Fig. 5 and Supplementary Figs. 9 and 10) to generate samples for analysis; M.J.O.W. developed methods and provided reagents; and L.R.S. and P.T.H. designed the study/experiments, developed methods, analyzed data and wrote the manuscript.

Corresponding authors

Correspondence to Len R Stephens or Phillip T Hawkins.

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The authors declare no competing financial interests.

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Supplementary Figures 1–10, Supplementary Tables 1–2, Supplementary Data 1–2 (PDF 901 kb)

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Clark, J., Anderson, K., Juvin, V. et al. Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry. Nat Methods 8, 267–272 (2011). https://doi.org/10.1038/nmeth.1564

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