Sequential Detection of Biomolecules in Formalin-fixed, Paraffin-embedded Samples with Mass Spectrometry Imaging.

Tissues are complex cellular environments, made up of a vast array of cell types and biomolecules all interacting with each other to carry out the functions of the organ. Traditionally, techniques for the analysis of biomolecules such as metabolites, glycans, and proteins involved the homogenization of tissue, destroying all spatial information. These traditional methods inhibited the complete understanding of complex intra- and extracellular molecular interactions. Mass Spectrometry Imaging (MSI), on the other hand, not only preserves the spatial information of biomolecules in tissue but also allows for multiple classes of analytes to be detected from the same tissue section through sequential analysis at a near-single-cell resolution. This enables us to derive a more complete picture of molecular interactions across different classes of biomolecules. The protocol presented here outlines the steps for performing mass spectrometry imaging of metabolites, N-linked glycans, and tryptic peptides sequentially from the same tissue section at a 20 µm resolution. Conscientious consideration of the order in which the classes of analytes are imaged, along with careful handling of the sections to ensure integrity, allows for multiple high-quality images to be collected from the same section. These data can subsequently be integrated with other spatial omics data (transcriptomics, immunohistochemistry, etc.) collected from serial sections, where the same cell can be analyzed in these adjacent sections.