Lipidview software

Phosphoinositide quantification was performed as described previously (Haag et al., 2012 (link)). Briefly, MDCK cells were plated in 6-well plates and grown for 5 days in Matrigel. Cells from two 6-well plates were combined and subjected to an acidic–neutral extraction of trichloroacetic acid (TCA)-washed cell pellets using PI(4)P 17:0–20:4 and PI(4,5)P₂ 17:0–20:4 from Avanti Polar Lipids as internal standards for quantification. Mass spectrometry was performed on a QTRAP 5500 instrument (AB Sciex) equipped with a Triversa NanoMate system (Advion Biosciences). Phosphoinositides were measured by scanning for neutral losses of m/z 357 (phosphatidylinositol) and m/z 437 (PIP₂) on an AB Sciex QTRAP 5500 instrument at collision energies of 25 eV and 35 eV, respectively. Mass spectra were evaluated using LipidView software (AB Sciex). PIP and PIP₂ amounts were normalized to total phospholipids, which was determined in neutral and acidic phases, as described previously (Özbalci et al., 2013 (link)).

Lipid extracts were directly infused into the TurboVion source by a syringe pump at 10 μl/min. and analysed by QTRAP5500 mass spectrometer (ABSCIEX, Farmington, MA, USA). Multiple precursor ion and neutral loss scanning methods were used for Information Dependent Acquisition of ms/ms data to detect and quantify the lipid classes as described earlier. Mass analyser conditions in the positive ion mode are as follows: Ionization Potential: 5500 V, Declustering Potential: 120 V, Entrance Potential: 9 V, Collision cell Exit Potential: 9 V. Collision energy for the survey scan was 10 and 45 eV for Enhanced Product Ion scans. In each scan, three ions with highest intensity were chosen for dependent product ion acquisition and the detected ions were excluded for the rest of the experiment after three occurrences. Data were analysed for the identification of lipid species using LipidView software (ABSCIEX). Lipid were quantified against internal standards and normalized against protein values obtained by the Bradford assay 19 (link).

Lipid extracts were fractionated on a Shimadzu ODS-3 C18 column at 40 °C and subjected to triple quadrupole mass spectrometry (QTRAP, AB SCIEX). Mass spectra were analyzed using LipidView software (AB SCIEX, US). Prior to the experimental runs, the spectrometry conditions were optimized based on the peak areas of a phosphatidylcholine standard. The fragmentation voltage was optimized by testing in the range 60-240 V in 20-V intervals, and the collision energy was set so that the strongest daughter ion response was 5-40 eV in second-order mass spectra. The lipid pathways were analyzed using a Lipid Pathway Enrichment Analysis (LIPEA) (https://lipea.biotec.tu-dresden.de/analyze), Biotechnology Center (BIOTEC). Technische Universität Dresden. Tatzberg 47/49. 01307, Dresden. Germany.

Qualitative analysis of shotgun-MS data was performed using the LipidView software (v2.0, ABSciex, Concord, ON, Canada). Software parameter settings: mass tolerance = 0.5, min % intensity = 1, minimum S/N = 10, flow injection average spectrum from top = 30% TIC, total double bonds ≤ 12.

All samples were measured 3 times in parallel. LipidView software (v2.0, ABSciex, Concord, ON, Canada) was used to undertake qualitative analysis of shotgun-MS data. In the process of data analysis, the relevant parameters of the software were set as follows: the mass tolerance was 0.5, the minimum% intensity was 1, the minimum signal-to-noise ratio was 10, the average flow injection spectrum from the top was 30% TIC, and the total double bond was ≤12. OriginPro (2021, OriginLab Corporation, Northampton, UK), SIMCA (14.1, Sartorius Lab Instruments GmbH & Co. KG, Goettingen, Germany), and Photoshop (2022, Adobe Systems Incorporated, San Jose, CA, USA) were used for plotting, data processing, and statistical analysis.