MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity using organic treatments.

Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CHCl3, hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30,000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophosphatidylinositol, confirming membrane lipid extraction from the tissues.