mass-spec-data-JCS-259145.zip

Mass spectrometry raw data from Article: https://doi.org/10.1242/jcs.259145AbstractTotal PtdInsP declines in cells overexpressing PI4KIIIα
Mass spectrometry was used to measure levels of PtdInsP, PtdInsP2, and PtdInsP3. Human tsA-201 cells were transiently transfected with PI4KIIIα, EFR3B, and TTC7B (PI4KIIIα), PIP5KIγ, or Dishevelled 3 (Dvl3). In cell populations overexpressing PIP5KIγ, we found as expected that PtdInsP, the substrate of the 5-kinase, declined by 29% and PtdInsP2, the product, increased by 41% compared to control cells. However, we were surprised to find that overexpression of PI4KIIIα reduced the prevalence of its immediate product, PtdInsP, by 22% while still increasing PtdInsP2 by 29%. PIP5KIγ also increases the prevalence of PtdInsP3 by 54%.
Dvl3 decreases PtdInsP and increases PtdInsP2 and PtdInsP3
We repeated these mass spectrometry measurements in cells overexpressing Dvl3 and found a 27% decrease in PtdInsP, a 21% increase in PtdInsP2, and a 66% increase in PtdInsP3.
MethodsMass spectrometry was performed as previously described (de la Cruz et al., 2020). In brief, lipids were precipitated from pelleted cells treated with trichloroacetic acid and extracted with chloroform/methanol. Dried extracts were methylated with TMS-DM and quantified by targeted analysis as described (Traynor-Kaplan et al., 2017). For each phosphoinositide type, peaks were analyzed for the three most predominant species: 38:4, 36:2, and 36:1. Peak areas were converted to milligrams per 106 cells as described using 37:4 lipid internal standards and DNA-per-sample measurements.
de la Cruz, L., A. Traynor-Kaplan, O. Vivas, B. Hille, and J.B. Jensen. 2020. Plasma membrane processes are differentially regulated by type I phosphatidylinositol phosphate 5-kinases and RASSF4. J Cell Sci. 133.
Traynor-Kaplan, A., M. Kruse, E.J. Dickson, G. Dai, O. Vivas, H. Yu, D. Whittington, and B. Hille. 2017. Fatty-acyl chain profiles of cellular phosphoinositides. Biochim Biophys Acta Mol Cell Biol Lipids. 1862:513-522.
Usage NotesMass
spectrometry data was acquired from a Waters Xevo TQ-S. Data are organized in
directories based on experimental day and each .raw folder contains the dataset
for a single sample. Excel files within each directory describe the
transfection conditions used for each sample: control (untransfected tsA-201
cells), PI4K (overexpressing PI4KIIIα, EFR3B, and TTC7B), PIP5K (overexpressing
PIP5KIγ), or Dvl (overexpressing Dishevelled 3). The amounts of
phosphoinositide standards are described as well.