Bioaffinity Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Direct Screening of Natural Product Extracts PDF Download

Author: Hoan Vu
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages :

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Book Description
Abstract: The search for new drugs in natural products involves the screening of natural product extracts on therapeutic targets for the presence of active compounds and subsequent investigation of their biological activities. Towards this end, a rapid and effective strategy to identify the noncovalent interactions between active ligands from natural product extracts with therapeutic protein targets without false positive or negative results is critical. The current thesis presents the outcomes of research exploring a novel approach of biological screening of natural product extracts using bioaffinity ESI-FTICR-MS as a detection means. A research plan containing three major steps has been undertaken in this study. Step one examines the current challenges in identifying protein complexes by ESI-MS and developing optimized conditions for ESI-FTICR-MS. Step two involves the development of methods for identifying complexes of targets with various molecular mass values (600 66,000 Da) in the setting of crude extracts. Step three uses these methods to screen natural product extracts and identify active natural product compound protein complexes, obtaining accurate molecular mass of active compounds and their mass fingerprint. On this basis, a mass-directed purification experiment was performed to isolate the identified active compounds. The binding of the pure active compounds with the protein target was then confirmed by competition experiments with a specific inhibitor of the protein. The procedure to achieve the optimal ESI-MS condition for detecting protein - small molecule complex has been successfully developed using bCA II as the test target protein and fractional factorial design (FFD) approach. Key instrumental factors controlling the desolvation process, as well as the interactions between these factors, have been identified using FFD experiment. It has been found that flow rate, nebulizer gas pressure and drying gas flow rate are factors that greatly affect the sensitivity of the complex detection. Capillary exit voltage also has a great effect on the desolvation in vacuum and on the preservation of the complex. These factors have been found to interact with each other and contribute to the final response, and therefore, have been incorporated in a second FFD experiment which focuses on minimum moment aberration. It has been found that the best condition is achieved when the capillary exit voltage is at the highest setting, and the flow rate, drying gas flow rate and nebulizing gas are at medium settings. The development of ESI-FTICR-MS methods for identifying noncovalent complexes in the environment of crude extracts has been accomplished using targets with various molecular mass values. In particular, ESI-FTICR-MS has been used to successfully detect hemin (