A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Abstract

Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP (18O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

Crucial Players for Inter-Organelle Communication: PI5P4Ks and Their Lipid Product PI-4,5-P2 Come to the Surface

“…Studies have shown that the levels of PI-5-P change in response to various stimuli such as insulin, oxidative stress, bacterial infection etc. and in turn regulate numerous cellular functions such as cell signaling, vesicular transport and even play a role in the nucleus (Hasegawa et al, 2017;Ghosh et al, 2019).…”

A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation

“…This method enables the quantification of phosphoinositide molecular species in a spectrum of biological samples, including those not amenable to radioisotope labeling. Ghosh et al have also described a mass spectrometry method based on the use of recombinant specific lipid-kinases to in vitro phosphorylate PI3P and PI5P into PI(3,5)P 2 and PI(4,5) P 2 , respectively 8 . However, it still remains a hurdle to simultaneously quantitate the acyl variants of all individual regioisomers of PI-monophosphate (PI3P/PI4P/PI5P) and -bisphosphate [PI (3,4)P 2 /PI(3,5)P 2 /PI(4,5)P 2 ] as well as PI and PI(3,4,5)P 3 in a biological sample 9 .…”

Septins tune lipid kinase activity and PI(4,5)P2 turnover during G-protein–coupled PLC signalling in vivo

“…The samples were dried, and reconstituted in 200 μl of methanol. LC MS/MS run method was used as described in Ghosh et al (2019) . The MRM transitions for 37:4 PI4P used was 995.5/613.5 and for 37:4 PI(4,5)P 2 was 1,103.5/613.5.…”