目前微藻的脂类分析主要侧重于脂肪酸酯、甘油三酯以及细胞内总脂的分析, 忽视了极性脂类的分析. 本研究建立了一个基于芯片的全自动电喷雾进样系统联用线性离子阱质谱的技术, 用于分析莱茵衣藻中的极性脂类. 通过数据库Lipidblast鉴定出了35种不同种类的极性脂类, 并首次鉴定出20种甜菜碱脂类. 同时利用了GC/TOF-MS来检测莱茵衣藻中的游离脂肪酸, 发现亚油酸、亚麻酸和棕榈酸是莱茵衣藻中含量最丰富的三种脂肪酸. 建立的脂类分析方法为微藻脂类种类全面综合的分析提供了方法上的参考.

Recently, microalgae as feedstock for lipid-based biofuel research has evoked great concern around the world. Previous lipid analyses focused on fatty acid esters, triglyceride and total lipid content in algae, but ignored the polar lipids analysis. Here, we present a comprehensive method to cover polar lipids and free fatty acids in Chlamydomonas reinhardtii. Firstly, lipid extraction was performed by methyl-tert-butyl ether which is well suited for shotgun lipidomics method. Afterwards, we developed a fast detection method in polar lipids analysis of C. reinhardtii by using a chip-based nanoelectrospray direct infusion with ion trap mass spectrometry (Nanomate-LTQ). A data-dependent MS/MS method acquired tandem mass spectra in positive and negative mode with a range of 10 minutes infusion time. MS/MS data obtained from Nanomate-LTQ were searched against automated MS/MS database with a precursor window of 0.4 Da and a product ion search window of 0.8 Da. Hit score represents the level of confidence from the Lipidblast library search which can be achieved by free availably NIST MS Search GUI. Therefore, thirty-five lipids were annotated by means of mass precursor search and subsequent in-silico MS/MS mass spectral database (Lipidblast) search, including most polar lipid species which are most common lipid species reported for C. reinhardtii. Among them, twenty classes of diacylglyceryltrimethylhomoserine that are very special lipid species in C. reinhardtii belonging to betaine lipids were annotated with in-house Lipidblast database. In order to validate this Lipidblast match, we performed a manual spectral interpretation of every experimental MS/MS spectrum to ensure authentic ester annotations. Besides, we also applied a gas chromatography tandem mass spectrometric method to detect free fatty acids in C. reinhardtii and found that linolenic acid, linoleic acid and palmitic acid are most abundant free fatty acids in C. reinhardtii. This study provides a potential comprehensive lipidomic method for further lipid analysis in algal research.