Histology-Directed Microwave Assisted Enzymatic Protein Digestion for MALDI MS Analysisof Mammalian Tissue

摘要

This study presents on-tissue proteolytic digestion using a microwave irradiation and peptide extraction method for in situ analysis of proteins from spatially defined regions of a tissue section. The methodology utilizes hydrogel discs (1 mm diameter) embedded with trypsin solution. The enzyme-laced hydrogel discs are applied to a tissue section, directing enzymatic digestion to a spatially confined area of the tissue. By applying microwave radiation, protein digestion is performed in 2 min on-tissue, and the extracted peptides are then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). The reliability and reproducibility of the microwave assisted hydrogel mediated on-tissue digestion is demonstrated by the comparison with other on-tissue digestion strategies, including comparisons with conventional heating and in-solution digestion. LC-MS/MS data were evaluated considering the number of identified proteins as well as the number of protein groups and distinct peptides. The results of this study demonstrate that rapid and reliable protein digestion canbe performed on a single thin tissue section while preserving therelationship between the molecular information obtained and the tissuearchitecture, and the resulting peptides can be extracted in sufficientabundance to permit analysis using LC-MS/MS. This approach will bemost useful for samples that have limited availability but are neededfor multiple analyses, especially for the correlation of proteomicsdata with histology and immunohistochemistry.