mass-spec-data-JCS-259145.zip

Mass spectrometry raw data from Article: https://doi.org/10.1242/jcs.259145Abstract<i>Total PtdInsP declines in cells overexpressing PI4KIIIα</i>Mass spectrometry was used to measure levels of PtdIns<i>P</i>, PtdIns<i>P</i>2, and PtdIns<i>P</i>3. 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 PtdIns<i>P</i>, the substrate of the 5-kinase, declined by 29% and PtdIns<i>P</i>2, 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, PtdIns<i>P</i>, by 22% while still increasing PtdIns<i>P</i>2 by 29%. PIP5KIγ also increases the prevalence of PtdIns<i>P</i>3 by 54%.<i>Dvl3 decreases PtdInsP and increases PtdInsP2</i> <i>and PtdInsP3</i>We repeated these mass spectrometry measurements in cells overexpressing Dvl3 and found a 27% decrease in PtdIns<i>P</i>, a 21% increase in PtdIns<i>P</i>2, and a 66% increase in PtdIns<i>P</i>3.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. <i>J Cell Sci</i>. 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. <i>Biochim Biophys Acta Mol Cell Biol Lipids</i>. 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.<br>