Enzyme Immobilized Microreactors And Surfactant Suppressed Matrix For Peptide Mapping And Low Mass Molecules Analysis With MALDI TOF-MS

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS) has become a rapidly burgeoning field in recent years. The advantages of high throughput, low detection limit, mixtures analysis ability as well as good tolerance to sample contamination make it powerful tool for biomolecular analysis.With fused silica capillary as support, immobilized enzyme microreactor has been prepared using covalent bonding or metal-ion chelating method. And a sensitive analytical procedure involving trypsin immobilized capillary microreactor for protein digestion, peptide mapping analysis with MALDI MS and internet database searching is then constructed for structure analysis and identification of minute amounts of proteins, and proved to work well even with as low as 9 finol protein sample.While kinetic parameters of enzymatic reaction are commonly determined by spectrophotometry, a simple and rapid means based on MALDI is proposed for characterization of the immobilized enzyme in the capillary microreactor. Using Na-benzoyloxycarbonyl-L-arginine as internal standard, the effects of the pH value of buffer and temperature on the activity and stability of the immobilized enzyme are investigated. The Michaelis-Menten constant of the immobilized enzyme, Km, is also determined.A new method based on the suppression of matrix signals with surfactant is developed for small molecular analysis, and the effect of concentration ratio of matrix to surfactant on the mass spectra is discussed. The reasonable mechanism and the detection limit of this method are also investigated. With this method, low molecular weight substances such as amino acids, peptides, drugs and cyclodextrins are analyzed successfully. And the feasibility of quantitative analysis of low-mass molecules by the developed method is also studied with suitable internal standard in samples.