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.

Key words: polar lipids, Chlamydomonas reinhardtii, direct infusion method, gas chromatography-mass spectrometry, free fatty acids