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On-tissue digestion has become the preferred method to identify proteins in mass spectrometry (MS) imaging. In this study, we report advances in data acquisition and protein identification for MS imaging after on-tissue digestion. Tryptic peptides in a coronal mouse brain section were measured at 50 μm pixel size and revealed detailed histological structures, e.g., the ependyma (consisting of one to two cell layers), which was confirmed by H&amp;E staining. This demonstrates that MS imaging of tryptic peptides at or close to cellular resolution is within reach. We also describe a detailed identification workflow which resulted in the identification of 99 proteins (with 435 corresponding peptides), based on comparison with LC-MS/MS data and in silico digest. These results were obtained with stringent parameters, including high mass accuracy in imaging mode (RSME &lt; 3 ppm) and at least two unique peptides per protein showing consistent spatial distribution. We identified almost 50% of proteins with at least four corresponding peptides. As there is no agreed approach for identification of proteins after on-tissue digestion yet, we discuss our workflow in detail and make the corresponding mass spectral data available as "open data" via ProteomeXchange (identifier PXD003172). With this, we would like to contribute to a more effective discussion and the development of new approaches for tryptic peptide identification in MS imaging. From an experimental point of view, we demonstrate the improvement due to the combination of high spatial resolution and high mass resolution/mass accuracy on a measurement at 25 μm pixel size in mouse cerebellum tissue. A whole body section of a mouse pub imaged at 50 μm pixel size (40 GB, 230,000 spectra) demonstrates the stability of our protocol. For this data set, we developed a workflow that is based on conversion to the common data format imzML and sequential application of freely available software tools. In combination, the presented results for spatial resolution, protein identification, and data processing constitute significant improvements for the field of on-tissue digestion. Graphical abstract MS imaging of coronal mouse brain cerebellum with a pixel size of 25 μm: A Optical image, B myelin staining, C H&amp;E staining, and D MS image overlay (RGB) of tryptic peptides m/z = 726.4045 ± 0.005, HGFLPR + H + (red), m/z = 536.3173 ± 0.005, AKPAK + Na + (green), and m/z = 994.5436 ± 0.005, WRQLIEK + Na + (blue).

Approaching cellular resolution and reliable identification in mass spectrometry imaging of tryptic peptides