An Investigation of High-throughput Proteomic Analysis in Marine Dinoflagellate (Symbiodiniaceae)

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The marine dinoflagellate in the family Symbiodiniaceae has received increasing attention due to its symbiotic interaction with reef-building corals. Integrity of coral reefs ecosystems is sustained through metabolic exchange between the two partners. However, this symbiosis can be disrupted (“dysbiosis”) under stress environment in response to the climate change. Despite intensive studies to understand the molecular mechanisms underpinning coral – Symbiodiniaceae interaction, the regulation mechanisms remain largely unknown. Recently, the advancement of bottom-up proteomic approaches and the influx of Symbiodiniaceae genetic information provide opportunities to understand the photoacclimation response for both free-living and in hospite Symbiodinium towards environmental stress. Therefore, this thesis applied high-throughput shotgun-based proteomics approaches to investigate and understand the molecular mechanism of light stress tolerance in marine dinoflagellate Symbiodinium. In this study, we compared filter - aided sample preparation (FASP), single-pot solid- phase-enhanced sample preparation (SP3), and stop-and-go-extraction tips (STAGETips, ST) based workflows to develop a high-throughput proteotyping protocol for Symbiodiniaceae algal research. Among the three techniques, SP3 provided the best performance with elevated detection of protein-peptides with the shortest operation time. To demonstrate the functionality of optimized SP3 workflow, this research successfully evaluated the proteome profile of Symbiodinium tridacnidorum, providing new insights into the light regulation mechanism. It appeared that light harvesting complex proteins as well as proteins linked to photosynthesis were upregulated when exposed under the low light condition. Further, we examined the proteome response of two Symbiodinium phylotypes: Breviolum sp. and Durusdinium sp. using isobaric tags for relative and absolute quantitation (iTRAQ) to assess their light stress responses. The proteomic analysis suggested the presence of unique light acclamation mechanisms in these two different Symbiodinium phototypes and showed the first characterisation of Symbiodinium proteomic profiles under two different light intensities. Finally, this thesis showed the first report on the Symbiodinium PCP extraction, purification and characterisation. Interestingly, we found that the purified PCP with IC50 values between 4–20 μM, exhibited significant anti-tumour and anti-inflammatory properties (against HTC-116 and MDA-MD 231 cancerous cell lines), which indicating novel bioactive compound for pharmaceutical industry use. Together, all these finding established SP3 proteomic-based as a high-throughput tool to understand the stress repones in Symbiodinium. Our proteomic investigation in Symbiodinium phylocaldes B and D suggested the first presence of unique light acclamation mechanism in two different Symbiodinium phototypes under low and hight light intensities. Lastly, the purified PCP presented as a promising candidate for pharmaceutical applications to cure chronic diseases.
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