Development of liquid microjunction extraction strategy for improving protein identification from tissue sections

MALDI Mass Spectrometry Imaging has shown important potential for molecular classification and pathology marker discovery. Protein markers identification is therefore of prime importance. Direct structural analysis from tissue sections has shown limitations for protein identification because of the high degree of complexity of tissues. Only proteins of major abundance are identified this way. On the contrary, conventional proteomics approaches clearly allow for reliable identification of complex protein extracts but do not provide fine correlation with protein location in their original context. Here is presented an approach to obtain identification of proteins of various abundances while keeping their localization within the section. On-tissue trypsin digestion followed by micro-extraction using a liquid micro-junction interface is an efficient strategy to extract tryptic peptides and further identify the associated proteins off tissues. It was shown that conventional Reverse Phase Liquid Chromatography separation on the extracted material followed by MS/MS analysis on a HR FTMS instrument enabled the identification of 1500 proteins on average with high confidence from an area of about 650μm in diameter, which corresponds to an estimated number of 1900 cells in average. The approach can be easily integrated in the MALDI MSI workflow and should provide interesting insights for clinical applications.