Computational protocol: Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection

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Protocol publication

[…] Slides for MALDI-MSI were removed from −80 °C and immediately desiccated for 30 min before matrix application. Alpha-cyano-4-hydroxycinnamic acid (CHCA, 5 mg/ml) in 50:50 acetonitrile: water and 0.2% trifluoroacetic acid was applied using an automatic matrix sprayer (TM-Sprayer, HTX Technologies) at 0.08 ml/min, 95 °C, with 6 passes. The MSI experiments were carried out using the ultrafleXtreme MALDI TOF/TOF instrument (Bruker Daltonics) equipped with a 2 kHz smartbeam laser. The raster width for imaging runs was 25–30 μm and 300 laser shots were collected at each raster position with a mass to charge ratio (m/z) range of approximately 100–1000. The imaging data was analysed using either FlexImaging 3.0 or 4.0 software (Bruker Daltonics) and normalised by total ion count. [...] DESI-MSI was performed on a Thermo Scientific Q-Exactive mass spectrometer equipped with an automated Prosolia 2D DESI source. A home-built DESI sprayer assembly was used with the spray tip positioned at 1.5 mm above the sample surface and at an angle of 75°. The distance between the sprayer to mass spectrometer inlet was 7 mm with a collection angle of 10°. The spray solvent was methanol/water (95:5 v/v), delivered at 1.5 μL/min using a Dionex Ultimate 3000 pump (Thermo Scientific) at a spray voltage of 4.5 kV. Nitrogen was used as the nebulisation gas at a pressure of 7 bars. The Q-Exactive mass spectrometer was operated in positive ion mode for all analysis using an S-Lens setting of 50 V. To acquire full mass spectra in the mass range of m/z 150–600 a mass resolution of 140000, AGC target of 5000000 and injection time of 200 ms was used. For acquisition of MS/MS spectra, a mass range of m/z 50–425 was used together with an injection time of 150 ms, AGC target of 1000000 and mass resolution of 35000. Fragmentation was performed using HCD setting of 30 and a mass isolation window of ± 0.3 Da. For distribution of the parent mass of m/z 400 a spatial resolution of 100 μm was used, while distribution of the fragment ion was acquired using 85 μm spatial resolution and corresponding scan speed was 130.21 μm/s and 472.22 μm/s respectively. Before analysis, samples were dried under a nitrogen stream for one minute, and full scan and fragmentation analysis was performed on adjacent tissue sections. For PalC standard analysis, PalC solubilized in DMSO was spotted on a slide and the spot was scanned to collect a spectrum. Data was converted into imzML format using imzML converter version 1.1.4.5 and data was visualised using MSIReader version 0.06. All intensities shown are raw ion intensities and 1st order linear interpolation was used for image generation. […]

Pipeline specifications

Software tools flexImaging, MSiReader
Application Mass spectrometry imaging
Organisms Salmonella enterica subsp. enterica serovar Typhimurium, Bacteria, Mus musculus
Diseases Gastroenteritis, Mastocytosis, Systemic
Chemicals Palmitoylcarnitine