title: Bioaffinity Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Direct Screening of Natural Product Extracts
Creators: name: Vu, Hoan
Publisher: name: Griffith University
Year: 2009
Subjects: subject: Natural product extracts
subject: Biological activities
subject: Electrospray ionization mass spectrometry
subject: Fourier transform spectroscopy

description: 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 (&lt; 1 kDa) and its complex with artemisinin. Accurate mass for artemisinin has been deduced with high resolution (deviation from theoretical value of less than 5 ppm). Screening 50 plant and marine sponge biota with hemin returned four active extracts. One of the four active natural product compounds had the molecular mass very close to artemisinin (Δ = 0.002 Da). The artemisinin identity of the active component has been confirmed using the MS2 pattern of artemisinin and the suspect artemisinin active component. A flavonoid was also detected in one of the active extracts and the structure elucidation of this active flavonoid has been proposed by comparing its diagnostic fragmentation patterns in multiple stage MS with those of other flavonones and flavonols. The first MS3 fragment ions of these 4 flavones and 2 flavonols are also presented in this study. The third and fourth active compounds have been isolated based on their accurate mass information using mass-directed purification. Their structures have also been determined by NMR. They are diacarnoic acids and are epi-isomers at position 3. MS2 experiments have been performed and can discriminate between these two compounds. Methods have also been developed for proteins having higher molecular masses using three target proteins ranging from 13 to 66 kDa. Using these methods, ESI-FTICR-MS or SEC-FTICR-MS have successfully identified these proteins and their complexes in a clean matrix, as well as in a natural product extract matrix. The use of microdialysis and SEC has been found to be a suitable method for protein desalting and buffer exchange, and for natural product extract filtering. The results support the conclusion that the ability of FTICR-MS to give accurate molecular mass of the inhibitors represents a powerful means for screening natural product extracts in a drug discovery process of finding diversified small molecule inhibitors. The methods developed for both direct infusion and online SEC-FTICR-MS have been successfully employed to screen 85 raw methanolic plant extracts with bCA II. A noncovalent complex has been detected and the active compound which binds to bCA II is identified. The specificity of the binding has also been confirmed by competition experiments with a specific inhibitor of the target protein. The outcomes of this work have been reported in the Journal of Biomolecular Screening (first published online on March 18, 2008). One of the key advantages of using ESI-FTICR-MS and associated methods is that the screening of the crude extracts is carried out without any preparation work such as pre-chromatographed, or pre-purification steps, thus avoiding any contamination which may lead to false positive or negative results. Information on the exact mass of the active compound also provides insights into the chemical composition or structural class of the compounds, thus shortening the long process of lead identification and allowing rapid mass-directed purification to isolate the active compounds. The demonstration of a coupled SEC-ESI-FTICR-MS procedure is superior to a batch method assay, whereby crude extracts are fractionated and subsequently bio-assayed until the active compound is found. Furthermore, this approach yields molecular masses in real time allowing subsequent mass-directed purification.