The application of mass spectrometry to Ginkgo biloba analysis and identification of phosphorylated proteins in response to elevated level of cCMP.

Mass spectrometry is widely used nowadays especially in the fields of pharmaceutical and proteomics research. Ginkgo biloba is one of the top selling phytophamiaceuticals in the US and Europe. The two major active components of Ginkgo leaf extract are the flavonoids and terpene lactones. Identification, determination, as well as the physiological effects of these two sets of compounds have been of increasing interest over the last 20 years. In this thesis, systematic qualitative and quantitative studies of the flavonoids and terpene lactones in Ginkgo biloba by liquid chromatography / mass spectrometry have been undertaken. Also in this thesis, mass spectrometric methodology was developed and applied to the identification of the proteins specifically phosphorylated in response to cCMP. Structural information of Ginkgo biloba flavonoids and terpene lactones, the fragment of compounds were obtained on both a LCQ ion trap and Q-TOF mass spectrometer. The tentative fragment pathways were proposed and used for structural elucidation of some unknown components in Ginkgo biloba commercial products. Capillary column separation of Ginkgo biloba commercial product was evaluated and fingerprint profiles of five Ginkgo biloba commercial products were compared. A reverse-phase high-performance liquid chromatography electrospray ionisation (RP-HPLC/ESI) mass spectrometry method was developed and validated for the simultaneous determination of ten major active components in Ginkgo biloba extract (bilobalide, ginkgolides A, B, C, quercetin, kaempferol, isorhamnetin, rutin hydrate, quercetin-3-beta-D-glucoside and quercitrin hydrate). The quantitative determination of flavonoids and terpene lactones by LC/MS in human urine after consumption of Ginkgo biloba product was developed. The online solid-phase extraction and capillary column with column-switch technique require minimum sample pre-treatment and both flavonoids and terpene lactones can be detected simultaneously. The mass accuracy at high molecular weight by matrix-assisted laser desoiption/ionisation time-of-flight mass spectrometry was investigated to resolve a question on mass accuracy which had been observed to be relatively low for high mss proteins. Bovine serum albumin (BSA) was employed as a model compound and strategies to improve mass measurement at high mass were examined. LC/MS was applied in part of the cyclic nucleotide project in the School of Biological Science. Since cAMP and cGMP are recognized second messengers and play important roles in signal transduction, to elucidate the function of cCMP in signal transduction, efforts were made to identify the cCMP-responsive protein kinase substrates. Methodology of specific enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) was developed, phosphorylated proteins responding specifically to cCMP were proposed, and this supports the relationship of cCMP with cell hyperproliferation