An optimized workflow for multiplexed and spatially localized on-tissue quantitative
protein analysis is here presented. The method is based on the use of an
enzyme delivery platform, a polymeric
hydrogel disc, allowing for a localized digestion directly onto the tissue surface coupled with an isobaric mass tag strategy for
peptide labeling and relative quantification. The digestion occurs within such
hydrogels, followed by
peptide solvent extraction and identification by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Since this is a histology-directed on-tissue analysis, multiple
hydrogels were placed onto morphologically and spatially different regions of interest (ROIs) within the tissue surface, e.g., cardiac
myxoma tumor vascularized region and the adjacent hypocellular area. After a microwave digestion step (2 min), enzymatically cleaved
peptides were labeled using TMT
reagents with isobaric mass tags, enabling analysis of multiple samples per experiment. Thus, N = 8
hydrogel-digested samples from cardiac
myxoma serial tissue sections (N = 4 from the vascularized ROIs and N = 4 from the adjacent hypocellular areas) were processed and then combined before a single LC-MS/MS analysis. Regulated
proteins from both cardiac
myxoma regions were assayed in a single experiment. Graphical abstract The workflow for histology-guided on-tissue localized protein digestion followed by isobaric mass tagging and LC-MS/MS analysis for
proteins quantification is here summarized.