Computational protocol: Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator

Similar protocols

Protocol publication

[…] Kidney tissues were obtained from 23 week-aged male diabetic Akita (C57BL/6J-Ins2Akita) mice and age-matched C57BL/6J control mice (n = 3/group). Snap frozen kidneys were quickly embedded in carboxymethyl cellulose (CMC) and sectioned (10 μm), and then thaw mounted onto an ITO glass slide. 9-AA (9-aminoacridine) (10 mg/mL in MeOH) was used as a MALDI matrix and applied by the SunCollect System (SunChrom, Friedrichsdorf, Germany). Just after matrix application, FTICR-MALDI-MSI was performed in negative ion mode using a SolariX MALDI-FTICR 7.0T Mass Spectrometer (Bruker Daltonics, MA) equipped with a Smartbeam II laser used at a repetition rate of 1 kHz. Mass spectra were acquired in the 100–700 m/z range in negative detection mode. The mass spectrum obtained for each position of the images corresponds to the averaged mass spectra of 300 consecutive laser shots on the same location with a time domain of 1MWord, subsequent single zero filling and sine wave apodization. An ICR noise threshold was fixed at 0.97 for imaging acquisition. Two image raster sizes were selected for whole kidney imaging at 70 μm and for high spatial resolution imaging of glomerular region at 30 μm. FTMSControl 3.0 and FlexImaging 3.2 software packages (Bruker Daltonics, Bremen, Germany) were used to control the mass spectrometer and set imaging parameters. The visualization and statistical analysis of imaging data were performed using MultImaging 1.0 software (ImaBiotech, Lille, France). For the detection of glomeruli, the localization of a phosphatidic acid (PA (36:1)) was used as a contrast ion as described previously by (, and regions of interest (ROIs) for glomeruli were drawn on the MS images. Fifteen glomeruli per animal were used for the analysis. For the determination of cortical tubular and medullary regions, phosphatidylinositol (PI (40:4)) and sulfatide (ST 42:1 (OH)) were used as contrast ions, respectively (). Periodic acid-Schiff (PAS) staining was performed on adjacent section of each fresh kidney tissue used for MALDI imaging acquisition and scanned using a 40 × magnification objective on an Olympus IX18 microscope (Olympus, Germany). Scans were then integrated in the MultImaging software for a co-registration with imaging data to accurately correlate the distribution of molecular species with kidney histological regions. […]

Pipeline specifications

Software tools flexImaging, Multimaging
Application Mass spectrometry imaging
Organisms Mus musculus
Diseases Diabetes Mellitus, Glucose Intolerance
Chemicals Adenosine Triphosphate, Ampicillin, Nucleotides, Lactic Acid