Brain Region-Specific Dynamics of On-Tissue Protein Digestion Using MALDI Mass Spectrometry Imaging

In mass spectrometry imaging (MSI), on-tissue proteolytic digestion is performed to access larger protein species and to assign protein identities through matching the detected peaks with those obtained by LC–MS/MS analyses of tissue extracts. The on-tissue proteolytic digestion also allows the analysis of proteins from formalin-fixed, paraffin-embedded tissues. For these reasons, on-tissue digestion-based MSI is frequently used in clinical investigations, for example, to determine changes in protein content and distribution associated with a disease. In this work, we sought to investigate the completeness and uniformity of the digestion in on-tissue digestion MSI. On the basis of an extensive experiment investigating three groups with varying incubation times: (i) 1.5 h, (ii) 3 h, and (iii) 18 h, we have found that longer incubation times improve the repeatability of the analyses. Furthermore, we discovered morphology-associated differences in the completeness of the proteolysis for short incubation times. These results support the notion that a more complete proteolysis allows better quantitation

Histology-Guided High-Resolution Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is widely used for clinical research because when combined with histopathological analysis the molecular signatures of specific cells/regions can be extracted from the often-complex histologies of pathological tissues. The ability of MSI to stratify patients according to disease, prognosis, and response is directly attributable to this cellular specificity. MSI developments are increasingly focused on further improving specificity, through higher spatial resolution to better localize the signals or higher mass resolution to better resolve molecular ions. Higher spatial/mass resolution leads to increased data size and longer data acquisition times. For clinical applications, which analyze large series of patient tissues, this poses a challenge to keep data load and acquisition time manageable. Here we report a new tool to perform histology guided MSI; instead of analyzing large parts of each tissue section the histology from adjacent tissue sections is used to focus the analysis on the areas of interest, e.g., comparable cell types in different patient tissues, thereby minimizing data acquisition time and data load. The histology tissue section is annotated and then automatically registered to the MSI-prepared tissue section; the registration transformation is then applied to the annotations, enabling them to be used to define the MSI measurement regions. Using a series of formalin-fixed, paraffin-embedded human myxoid liposarcoma tissues, we demonstrate an 80% reduction of data load and acquisition time, thereby enabling high resolution (mass or spatial) to be more readily applied to clinical research. The software is freely available for download

Multimodal Mass Spectrometry Imaging of <i>N</i>‑Glycans and Proteins from the Same Tissue Section

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the <i>in situ</i> multimodal analysis of <i>N</i>-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both <i>N</i>-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique <i>N</i>-glycan and peptide identities