Site‐to‐Site Reproducibility and Spatial Resolution in MALDI–MSI of Peptides from Formalin‐Fixed Paraffin‐Embedded Samples

Purpose To facilitate the transition of MALDI–MS Imaging (MALDI–MSI) from basic science to clinical application, it is necessary to analyze formalin‐fixed paraffin‐embedded (FFPE) tissues. The aim is to improve in situ tryptic digestion for MALDI–MSI of FFPE samples and determine if similar results would be reproducible if obtained from different sites. Experimental Design FFPE tissues (mouse intestine, human ovarian teratoma, tissue microarray of tumor entities sampled from three different sites) are prepared for MALDI–MSI. Samples are coated with trypsin using an automated sprayer then incubated using deliquescence to maintain a stable humid environment. After digestion, samples are sprayed with CHCA using the same spraying device and analyzed with a rapifleX MALDI Tissuetyper at 50 µm spatial resolution. Data are analyzed using flexImaging, SCiLS, and R. Results Trypsin application and digestion are identified as sources of variation and loss of spatial resolution in the MALDI–MSI of FFPE samples. Using the described workflow, it is possible to discriminate discrete histological features in different tissues and enabled different sites to generate images of similar quality when assessed by spatial segmentation and PCA. Conclusions and Clinical Relevance Spatial resolution and site‐to‐site reproducibility can be maintained by adhering to a standardized MALDI–MSI workflow.