Identification of proteins directly from tissue: in situ tryptic digestions coupled with imaging mass spectrometry

A novel method for on-tissue identification of proteins in spatially discrete regions is described using tryptic digestion followed by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with MS/MS analysis. IMS is first used to reveal the protein and peptide spatial distribution in a tissue section and then a serial section is robotically spotted with small volumes of trypsin solution to carry out in situ protease digestion. After hydrolysis, 2,5-Dihydroxybenzoic acid (DHB) matrix solution is applied to the digested spots, with subsequent analysis by IMS to reveal the spatial distribution of the various tryptic fragments. Sequence determination of the tryptic fragments is performed using on-tissue MALDI MS/MS analysis directly from the individual digest spots. This protocol enables protein identification directly from tissue while preserving the spatial integrity of the tissue sample. The procedure is demonstrated with the identification of several proteins in the coronal sections of a rat brain.