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.
Highlights
Enzymes immobilized on various support materials are frequently used for their improved enzyme stability, activity, and possibility to effectively control an enzymatic reaction
The proteolytic activity of enzymes trypsin, α-chymotrypsin, and subtilisin A immobilized on indium tin oxide (ITO) glass by ambient ion soft landing was tested by in situ holomyoglobin digestion performed at two different reaction times
Current techniques for protein coupling to solid substrates are based on formation of a covalent bond between enzyme and stationary phase
Summary
Enzymes immobilized on various support materials are frequently used for their improved enzyme stability, activity, and possibility to effectively control an enzymatic reaction. Immobilization of enzymes by ion soft landing was reported to be an appropriate technique for in situ digestion of proteins [3]. Ion soft landing refers to the process where the hyperthermal molecular ions are nondestructively captured on conductive target supports [4]. This approach was first described by Cooks in 1977. Applications of limited proteolysis and mass spectrometry were reported for antibody epitope mapping [25,26], mapping of protein–DNA interaction [27], or characterization of protein structural changes in a whole yeast cell proteome [28]. This study represents a novel approach for monitoring alterations in protein tertiary structure, which combines in situ limited proteolysis on functionalized chips with MALDI mass spectrometry
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