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A simultaneous on-tissue proteolytic digestion and extraction method is described for the in-situ analysis of proteins from spatially distinct areas of a tissue section. The digestion occurs on-tissue within a hydrogel network, and peptides extracted from this gel are identified with liquid chromatography tandem MS (LC-MS/MS). The hydrogels are compatible with solubility agents (e.g. chaotropes and detergents) known to improve enzymatic digestion of proteins. Additionally, digestions anddoi:10.1021/ac3031493 pmid:23402265 pmcid:PMC3595595 fatcat:wgq4cj67o5arhnqxtuhu7ckgb4