Integrated Mass Spectrometry Imaging and Omics Workflows on the Same Tissue Section Using Parafilm-Assisted Microdissection

In this work, we demonstrate the use of grid-aided, parafilm-assisted microdissection to perform MALDI MS imaging and shotgun proteomics and metabolomics in a combined workflow and using only a single tissue section. The grid is generated by microspotting of acid dye 25 using a piezoelectric microspotter. In the gas phase, the dye is detectable as a free radical species, and its distribution can be superimposed with ion images generated by tissue components, then used as a guide to locate regions of interest and aid during manual microdissection. Subjecting the dissected pieces to the modified Folch method allows to separate the metabolic components from the proteins. The proteins can then be subjected to overnight digestion under controlled conditions to improve protein identification yields. The proof of concept experiment on rat brain generated 162 and 140 metabolite assignments from three ROIs (cerebellum, hippocampus and midbrain/hypothalamus) in positive and negative mode, respectively, and 890, 1,303 and 1,059 unique protein accessions. Integrated metabolite and protein overrepresentation analysis identified pathways associated with the biological functions of each ROI, most of which were not identified when looking at the protein and metabolite lists individually. This combined MALDI MS imaging and multi-omics approach benefits from the advantages of both methods (molecular mapping from MSI and increased depth of coverage from shotgun proteomics and metabolomics), and further extends the amount of information that can be generated from single tissue sections.