Scils lab mvs 2018b

MSI was performed in positive ion reflector mode over a mass range of m/z 600–3200 on rapifleX MALDI Tissuetyper TOF mass spectrometers (Bruker) fitted with a Smartbeam 3D Nd:YAG (355mm) laser. The Smartbeam parameter was set to M5 small, with the Imaging 50 µm application, 15 µm scan range, and resulting field size of 50 µm. Spectra were accumulated from 500 laser shots at 10 kHz frequency with a sampling rate of 1.25 GS s^–1 and baseline subtraction performed during acquisition. The “Detector check” function was conducted regularly at each site in order to maintain this variable setting at appropriate voltages. For every measurement, the instruments were externally calibrated using the Peptide Calibration Standard II (Bruker),12 laser power adjusted according to on‐tissue test shots in order to reach the optimal ionization threshold, and two non‐tissue measurement regions included. Following MALDI–MSI, the matrix was eluted from the samples with 70% ethanol, stained with hematoxylin and eosin (H&E) and scanned with 20× objective magnification on a NanoZoomer SQ (Hamamatsu Photonics Deutschland, Munich, Germany), Aperio AT (Leica Biosystems, Buffalo Grove, IL, USA), or Mirax Desk slide scanner (Carl Zeiss MicroImaging, Göttingen, Germany). The H&E image was then co‐registered to the respective measurement in flexImaging v. 5.0 or SCiLS Lab MVS 2018b software (Bruker).

MALDI-MSI experiments were carried out in positive ion reflectron mode over a mass range of m/z 100 to 1000 using a MALDI rapifleX Tissuetyper MALDITOF/TOF MS (Bruker Daltonics, Bremen, Germany) equipped with a 10 kHz Smartbeam 3DTM Nd:YAG laser. Data collected on the rapifleX was at a spatial resolution of 50 µm, summing up 400 laser shots/raster position. FlexImaging 5.0 (Bruker Daltonics, Bremen, Germany) software was used for initial data analysis. Normalization, molecular image extraction, and regions of interest were defined in SCiLS Lab MVS 2018b (Bruker Daltonics, Bremen, Germany) software typically using a mass selection window of ± 0.05 Da. AZD2858 was detected as the protonated molecular ion (M + H+) at m/z 454.2.

A stock solution of 3M-4-TMAB and 4-TMAP (AstraZeneca compound management) was prepared daily as needed at a concentration of 1 mg/ml in 50:50 (v/v) methanol/water. The stock solutions (1 mg/ml) were subsequently mixed to form a working solution (0.1 mg/ml), which was then serially diluted to 11 further working solutions with concentrations in the range of 0.00002 to 0.04 mg/ml. Working solutions at 0.00002, 0.00004, 0.0001, 0.0002, 0.0004, and 0.001 mg/ml were spotted (50 nl using a Biospotter, Biofluidix, Freiburg, Germany) onto GF mouse brain tissue and allowed to evaporate. This deposition of standard translated to 0.001, 0.002, 0.005, 0.01, 0.02, and 0.05 ng of 3M-4-TMAB/4-TMAP on tissue. DESI-MSI analysis was performed using the same parameters as above. Data visualization and region of interest extraction were performed using SCiLS Lab MVS 2018b (Bruker Daltonics, Bremen, Germany) software typically using mass selection window of ±0.05 Da. 3M-4-TMAB/4-TMAP was detected as the protonated molecular ion (M⁺H⁺) at m/z 160.1. Extracted mean relative abundance was used from each area of interest to construct calibration curves in Microsoft Excel using a linear fit with unknown endogenous concentrations calculated using the equation of a straight line.