Application & Background
The need for consistent, homogeneous MALDI matrix deposition is crucial for imaging studies that span multiple analyses. This may be true for imaging studies with large numbers of subjects, timepoints, or oversized samples that do not fit onto a single target plate (and will require multiple MS analyses with subsequent stitching of data; e.g., whole-rat sections). Uncontrolled variations in the matrix coating step of an imaging experiment can adversely affect the extraction efficiency of your analytes of interest, as well as, affect the imaging parameters of the experiment such as laser power and number of laser shots to accumulate for each acquisition. These variations can make comparisons of imaging data produced from successive experiments challenging, and in the case of oversized samples, unnecessarily difficult to define normalization factors in order to make the images comparable and amenable to stitching.
Here we describe a robust and reproducible MALDI matrix coating protocol using the TM-Sprayer for the preparation of oversized whole-rat tissue sections.
Optical image of whole-rat section after completion of the MALDI spray coating using the optimized HTX TM-Sprayer protocol described herein.
Experimental
experimental design
A single 10 mg/kg PO dose of Olanzapine was administered to a male Sprague-Dawley rat, euthanized at 6 hours post-dose and flash frozen.
The whole-animal carcass was sectioned (20 μm thickness) and sagittal whole-body sections were transferred to MALDI target plates using double-sided tape.
Matrix Application using the HTX TM Sprayer
Tissue sections were then sprayed with 2,5-Dihydroxbenzoic (DHB) matrix (40mg/ml, 70/30 Methanol/H2O spiked with 2 uM IS) using the HTX TM-Sprayer and the following conditions shown in Table 1.
Table 1. Spraying parameters for matrix deposition using the HTX TM Sprayer.
MALDI MSI Data collection
Images were collected across the entire tissue area at 500 μm pixel resolution using a 7.0T SolariX FTMS system (Bruker Daltonics) equipped with a dual ESI-MALDI source employing smartbeam-II™ technology. The laser was operated at 1 kHz and a total of 500 laser shots were accumulated from each pixel position. Data were collected in full scan mode over a mass range of m/z 100 to 1500.
Full scan data were processed and drug and metabolite images were extracted and displayed using FlexImaging software 3.0 (Bruker).
results
High-resolution FTMS data were imported into FlexImaging for processing and ion image extraction. To assess overall matrix coverage and image performance, the ion image representing the matrix-spiked internal standard was extracted (Figure 1). It can be seen that the non-tissue regions (along the outer edge of the tissue section) provided the highest signal intensities. There also appears to be regions or specific organs of the whole-rat tissue where the IS signal has been suppressed. Representative pixels from the top, middle, and bottom of the non-tissue regions were selected and spectra were compared for peak intensity, resolution, and signal to noise (Figure 2). Since the IS response proved consistent throughout the run, subsequent olanzapine and metabolite ion images were extracted and stitched, allowing for the visualization of analyte distributions across a whole-rat section in the proper orientation (Figures 3-5).
Figure 1. MALDI MS Image of internal standard.
Figure 3. MALDI MS Image of Olanzapine (stitched) (313.1517 m/z)
Figure 4. MALDI MS Image of Oxidative Metabolite (stitched) (329.1484 m/z)
Figure 5. MALDI MS Image of N-desmethyl Metabolite (stitched) (299.1356 m/z)
The tissue images and MS data presented in this note were provided by Dr. Sheerin Shahidi-Latham and Cristine Quiason, Genentech Inc. South San Francisco, CA, USA.