DOI: 10.6084/m9.figshare.24309139.v1
title: Mass spectral analysis from untargeted lipidomics of primary murine bone marrow-derived macrophages infected with Legionella pneumophila

description: The excel file contains LC-MS spectral count dataset for lipids isolated from murine BMMs that were either (i) uninfected, (ii) treated with BSA alone (iii) infected with Legionella pneumophila or (iv) infected with Legionella pneumophila and treated with 150µM palmitoleic acid complexed to BSA. This is a source file associated with the following manuscript: <b>"The intracellular growth of the vacuolar pathogen </b><b><i>Legionella pneumophila</i></b><b> is dependent on the acyl chain composition of the host membranes."</b>Ashley A. Wilkins<sup>1</sup>, Benjamin Schwarz<sup>2</sup>, Ascencion Torres-Escobar<sup>1</sup>, Reneau Castore<sup>1,3</sup>, Layne Landry<sup>4</sup>, Brian Latimer<sup>5</sup>, Eric Bohrnsen<sup>2</sup>, Catharine M. Bosio<sup>2</sup>, Ana-Maria Dragoi<sup>3,5</sup>, Stanimir S. Ivanov<sup>1*</sup><sup>1</sup>Department of Microbiology and Immunology, LSU Health Shreveport, Shreveport, LA, USA<sup>2</sup>Research and Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, 59840, USA<sup>3</sup>Department of Molecular and Cellular Physiology, LSU Health Shreveport, Shreveport, LA, USA<sup>4</sup>School of Medicine, LSU Health Shreveport, Shreveport, LA, USA<sup>5</sup>Innovative North Louisiana Experimental Therapeutic (INLET) Program, Feist-Weiller Cancer Center, LSU Health Shreveport, Shreveport, LA, USA<b>* Correspondence:</b><br>Stanimir Ivanov, Ph.D.<br>stanimir.ivanov@lsuhs.edu ----- Pertinent material and methods information ----<b>Cellular infections for lipidomics analysis:</b> BMMs were seeded in 6 well tissue culture plates (2X10<sup>6 </sup>cells/well) in re-plating media (RPMI 1640 with L-glutamine, 10�S, 10% M-CSF-conditioned media) for 2hrs followed by 14hr incubation with serum-free RPMI (SF-RPMI). Cells were infected with liquid culture-grown <i>Lp01 ∆flaA</i> at MOI=5 for 8hrs in SF-RPMI. Plates were centrifuged for 5 min at 800rpms to increase bacteria attachment to host cells immediately after the inoculum was added. Palmitoleic acid complexed with BSA (200µM final concentration) was added at 2hpi in SF-RPMI. Each infection condition in every experiment was carried out in two technical replicates, which were pooled together after sample preparation. Samples from eight biological repeats were prepared for LCMS analysis as detailed below.<b>Metabolite and Lipid Sample Preparation: </b>For all LCMS methods LCMS grade solvents were used. Media was removed and discarded from cell culture samples in 6 well plates and washed briefly with 1 mL of 0.9 % sodium chloride. Wash was gently removed, and 0.4 mL of ice-cold methanol was added to each well. Plates were placed on ice for 5 min. To each sample 0.4 mL of water was added and the well was scraped to suspend cell-associated solids. Samples were transferred to microtubes and 0.4 mL of chloroform was added to each. Samples were agitated for 30 minutes at 4<sup>°</sup>C and subsequently centrifuged at 16k xg for 20 min to induce layering. An 0.4 mL aliquot of the organic layer (bottom) was dried in a Savant SpeedVac SPD130 (Thermo Scientific) and resuspended in 1 mL of 5 µg/mL butylated hydroxytoluene in 6:1 isopropanol:methanol.<b>Liquid Chromatography Tandem Mass Spectrometry: </b>LCMS grade water, methanol, isopropanol and acetic acid were purchased through Fisher Scientific.<b> </b>Bulk lipids were analyzed as previously described (REFs Schwarz, Foliaki). A Shimadzu Nexera LC-20ADXR was used for chromatography across a Waters XBridge® Amide column (3.5 µm, 3 mm X 100 mm) with a 12-minute binary gradient from 100% 5mM ammonium acetate, 5% water in acetonitrile apparent pH 8.4 to 95% 5mM ammonium acetate, 50% water in acetonitrile apparent pH 8.0 was used to separate organic fraction samples. Lipids were detected using a Sciex 6500+ QTRAP® mass spectrometer with polarity flipping. Lipids were detected using scheduled MRMs based on acyl chain product ions in negative mode or a combination of invariant or neutral loss combinations in positive mode.<b> </b>All signals were integrated using MultiQuant® Software 3.0.3. Signals with greater than 50% missing values were discarded and remaining missing values were replaced with the lowest registered signal value. All signals with a QC coefficient of variance greater than 40% were discarded. Filtered datasets were total sum normalized prior to analysis. Single and multi-variate analysis was performed in MarkerView® Software 1.3.1. Datasets were z-scaled for display across the displayed groups. Z-score sums were calculated by summing all lipid signals with a given lipid characteristic in the z-scaled dataset that pass a statistical criterion. The entire lipidomics data set is available at the Figshare data repository