Proteases immobilization for in situ time-limited proteolysis on MALDI chips

Abstract

A large number of different enzyme immobilization techniques are used in the field of life sciences, clinical diagnostics, or biotechnology. Most of them are based on a chemically mediated formation of covalent bond between an enzyme and support material. The covalent bond formation is usually associated with changes of the enzymes’ three-dimensional structure that can lead to reduction of enzyme activity. The present work demonstrates a potential of an ambient ion-landing technique to effectively immobilize enzymes on conductive supports for direct matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analyses of reaction products. Ambient ion landing is an electrospray-based technique allowing strong and stable noncovalent and nondestructive enzyme deposition onto conductive supports. Three serine proteolytic enzymes including trypsin, α-chymotrypsin, and subtilisin A were immobilized onto conductive indium tin oxide glass slides compatible with MALDI mass spectrometry. The functionalized MALDI chips were used for in situ time-limited proteolysis of proteins and protein–ligand complexes to monitor their structural changes under different conditions. The data from limited proteolysis using MALDI chips fits to known or predicted protein structures. The results show that functionalized MALDI chips are sensitive, robust, and fast and might be automated for general use in the field of structural biology.