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Jahreis, Bastian ; Kokesch-Himmelreich, Julia ; Race, Alan M. ; Römpp, Andreas:
Towards Cellular Resolution of Tryptic Peptides in Tissue Sections by MALDI MS Imaging : A Focus on Enzyme Application and Reproducibility.
In: Analytical Chemistry.
(Juli 2026)
.
ISSN 1520-6882
DOI: https://doi.org/10.1021/acs.analchem.6c01111
Angaben zu Projekten
| Projekttitel: |
Offizieller Projekttitel Projekt-ID SFB 1357: MIKROPLASTIK – Gesetzmäßigkeiten der Bildung, des Transports, des physikalisch-chemischen Verhaltens sowie der biologischen Effekte: Von Modell- zu komplexen Systemen als Grundlage neuer Lösungsansätze 391977956 |
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| Projektfinanzierung: |
Deutsche Forschungsgemeinschaft |
Abstract
Bottom-up proteomics coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI imaging) is the preferred method for untargeted localization and identification of proteins in tissue. However, there are no MALDI imaging workflows that combine high lateral resolution, accurate mass detection, efficient tryptic digestion, and reproducibility. In this study, we developed a MALDI imaging workflow that enables reproducible imaging of tryptic peptides in mammalian tissue sections with high resolution in mass and space. For the first time, tryptic peptides could be imaged in tissue sections at 10 μm pixel size with high mass resolution (R > 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.