Towards Cellular Resolution of Tryptic Peptides in Tissue Sections by MALDI MS Imaging: A Focus on Enzyme Application and Reproducibility

> 100,000 FWHM), mass accuracy (RMSE < 3 ppm), and without discernible delocalization. We achieved this by systematic optimization of trypsin spray parameters, including the number of sprays, enzyme volume, density, flow rate, and a custom-built digestion chamber specifically designed for high-resolution measurements. MALDI imaging at a 10 μm pixel size showed that multiple (10) sprays followed by short incubations and balanced on-tissue moisture are crucial to avoid delocalization and obtain highly resolved peptide images with sufficient ion intensities. As a result, up to 283 proteins could be identified using our optimized method based on additional LC-MS/MS data and strict identification criteria. Increased peptide intensity enabled on-tissue MALDI-MS/MS of 16 tryptic peptides and confirmed their amino acid sequence. Our results provide insights into the complex on-tissue digestion dynamics associated with MS imaging. Although aimed at high spatial resolution, many findings can be transferred to other MALDI imaging workflows to make it suitable for different spatial proteomics applications.