Analysis The Phospholipid And Cholesterol Ester By Shotgun Lipidomics And Its Application

There are three parts in this paper. The first part was establishing the method for analyzing phospholipids (PL) and cholesterol ester (CE) by direct injection electrospray ionization tandem mass spectrometry (DI-ESI-MS/MS) and flow injection electrospray ionization tandem mass spectrometry (FI-ESI-MS/MS), respectively. Using the method, the profile of PL and CE from some biological samples was analyzed. The second part of the research was tracking PL profiling from Ctenopharyngodon idellus muscle during storage at different temperatures. The third part of the paper was investigating the effect of enrofloxacin (ENR) to PL and CE profiling from Carassius auratus liver by shotgun lipidomics analysis method.Firstly, the methods for analyzing PL by DI-ESI-MS/MS and for analyzing CE by FI-ESI-MS/MS were established. Lipids were extracted with a modified Bligh & Dyer method, and then the analysis was taken directly into ESI source by injection pump or flow injection system. The MS/MS experiments combining precursor ion scan and neutral loss scan were performed to monitor different classes of PL (phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, phosphatidyl glycerol and phosphatidic acid) and CE by tandem triple quadrupole mass spectrometry. Methodological evaluation showed that the intensity of quasi-molecular ion of PL and CE to its concentration were in good linear relation in a certain range. The recovery and precision of the methods meet the requirements for analyzing biological samples. The research strategy of shotgun lipidomics was simple, fast and accurate for qualitative and quantitative, with excellent stability and reproducibility, thus making it would be suitable to analyze PL and CE from various biological samples for lipidomics research.Then we analyzed the profiling of PL and CE from some biological samples. For one hand, the profiling of PL from two species of freshwater fish (C. idellus and C. auratus) and three species of marine fish (Trichiurus haumela, Pampus argenteus and Inimicus japonicus) muscle were analyzed. The results showed that the molecular species and the content of PL between the freshwater and marine fish are different. The content of lyso-phospholipids (LPL) and polyene PL from freshwater fish is less than marine fish, and the fatty acid chain constituents of polyene i’l from marine fish mainly are polyunsaturated fatty acid, especially ARA, EPA and DHA. For the biological activity and nutritional value of the fish polyene PL are higher than that from soybeans, thus it may have a high application potential. For another hand, the profiling of CE from egg yolk and crab (Scylla serrata) yolk were analyzed by FI-ESI-MS/MS. The results showed that CE profile between egg yolk and crab yolk are different. The CE from egg yolk mainly is even carbon atoms fatty acid, while there are some odd carbon atoms CE molecular species in crab yolk. The content of saturated and polyunsaturated cholesterol esters from egg yolk are less than the crab yolk, while the content of mono-unsaturated cholesterol ester is higher than the crab yolk. The molecular species of polyunsaturated CE from egg yolk mainly are CE 18:2, while for crab yolk mainly is CE 22:6, CE 20:5 and CE 20:4.Subsequently, we investigated the PL profiling of muscle from C. idellus during storage at different temperatures (+20℃,+4℃and -20℃) for different time (72 h,3 day and 90 day). The result showed that the oxidation and hydrolysis are the two main causes for the deterioration of PL storage at +20℃and +4℃. The most content of PL molecular species increased and then decreased gradually. The deterioration of PL storage at +20℃is much faster than that at +4℃. However, some special PE molecular species with low abundance formerly, such as PE 16:0/16:1, PE 16:1/18:1 and PE 16:0/18:1, emerged during the storage in quantity. It indicated that those PE molecular species may come from the microbe bred in the muscle and its content may be expected to become a new parameter for evaluation the quality of fish. The molecular species and content of PL from C. idellus muscle storage at -20℃for 90 day did not have significantly change. It indicated that the PL could stay stability during low temperature storage for a long time.Finally, we discussed the effect of ENR to PL and CE profiling from C. auratus liver by shotgun lipidomics analysis method. The results show that low concentrations of ENR would affect the membrane lipid composition and content from C. auratus liver, especially the content decrease in PL and the increase in LPL and CE. With the reduction of ENR by metabolic conversion, the membrane lipids returned to normal gradually. It indicated that the damage affecting from hepatic lipid metabolism and membrane system of fish is limited at low concentrations of ENR in short-term. If ENR doses were controlled reasonably, the harmful to fish could be reduced in minimally. While increase the dosage or overusing, the effects of ENR to the liver will not be only the slight biochemical changes, but liver failure may occur and lead fish to deaths.