Localized in situ hydrogel-mediated protein digestion and extraction technique for on-tissue analysis.

Harris GA, Nicklay JJ, Caprioli RM
Anal Chem. 2013 85 (5): 2717-23

PMID: 23402265 · PMCID: PMC3595595 · DOI:10.1021/ac3031493

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 and extractions are compatible with imaging mass spectrometry (IMS) experiments. As an example application, an initial IMS experiment was conducted to profile lipid species using a traveling wave ion mobility mass spectrometer. On-tissue MS/MS was also performed on the same tissue section to identify lipid ions that showed spatial differences. Subsequently, the section underwent an on-tissue hydrogel digestion to reveal 96 proteins that colocalized to the rat brain cerebellum. Hematoxylin and eosin (H & E) staining was then performed to provide additional histological information about the tissue structure. This technology provides a versatile workflow that can be used to correlate multiple complementary analytical approaches in the analysis of a single tissue section.

MeSH Terms (12)

Animals Cerebrum Chemical Fractionation Chromatography, Liquid Hydrogels Molecular Weight Proteins Proteolysis Rats Rats, Sprague-Dawley Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Tandem Mass Spectrometry

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