Development and Application of Immobilized Enzyme Reactors Coupled with Nanoelectrospray Mass Spectrometry in Proteomics
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Nanoelectrospray ionization (nanoESI) is a highly efficient method for direct analysis in mass spectrometry with better sensitivity toward a variety of peptide and protein analyses compared to electrospray ionization (ESI). Enzyme immobilization is a fast-growing application in proteomics because of its robustness to simplify processing steps with a faster rate. Immobilized enzymes enable longer duration of activity of enzymes and repeat uses of enzyme with high activity, high selectivity, and high stability. This dissertation provided the kinetic study of the immobilized trypsin microreactor (IMTR) coupled with nanoESI mass spectrometry developed in our lab. The results showed the immobilized trypsin's intrinsic kinetic behaviors are different compared to the free trypsin's, and immobilized trypsin retains good stability and high biocatalytic activity in aqueous/organic solvent mixtures. Additionally, the immobilized pepsin microreactor (IMPR) coupled with nanoESI mass spectrometry was developed in this dissertation. The application of the immobilized pepsin microreactor in hydrogen/deuterium (H/D) exchange was studied. The results showed the immobilized pepsin microreactor coupled with H/D exchange mass spectrometry is rapid, completes the digestion of protein, and alleviates the problem of serious back exchange in the process of in-solution digestion and HPLC separation. In general, immobilized enzyme microreactor coupled with nanoESI mass spectrometry has been demonstrated to be a good tool for online digestion of proteins in proteomics.