High-Resolution Liquid Chromatography-Mass Spectrometry for Lipidomics.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a powerful tool for identification and classification of lipids. Ultra-high performance liquid chromatography (UHPLC) allows for robust separations of complex mixtures, while high-resolution mass spectrometry (HRMS) identifies compounds with efficiency and accuracy (Zullig T and Kofeler HC, Mass Spectrom Rev 40:162-176, 2021). The high specificity and sensitivity of mass spectrometry makes it the method of choice when analyzing lipids (Kofeler HC, J Lipid Res 62:100138, 2021). Untargeted mass spectrometry identifies all lipids within a sample and is useful for identification and further discovery. This chapter describes the use of a Q Exactive mass spectrometer to perform an untargeted LC-MS/MS lipidomics analysis.

Analysis of Cholesterol Lipids Using Gas Chromatography Mass Spectrometry.

An optimized Bligh and Dyer protocol and subsequent derivatization is described in this chapter for the extraction of free cholesterol and cholesterol esters from tissue samples. Quantification analysis of lipid species is then described utilizing gas chromatography-mass spectrometry, the ideal method for analysis of volatile organic compounds and extraction of sterols.

Analyses of pseudoexfoliation aqueous humor lipidome.

Pseudoexfoliation syndrome (PEX) is a systemic disorder that manifests as a fluffy, proteinaceous fibrillar material throughout the body. In the eye, such deposits result in glaucoma (PEXG), due to impeding aqueous humor outflow. Serum lipid alterations and increased lipid peroxidation have been reported in PEX. We report the first ever comprehensive lipid profiling of the aqueous humor (AH) of PEXG. Our untargeted lipidomic analysis of 23 control, 19 primary open angle glaucoma (POAG), 9 PEX, and 14 PEXG AH patients resulted in the identification of 489 lipid species within 26 lipid classes across PEX, PEXG, POAG, and control AH samples. Multiple cholesterol esters (ChEs), phosphatidylcholines (PCs), triglycerides (TGs), and ceramides (Cers) were present in higher concentrations in the PEXG AH than in all other groups. CerG2GNAc1(d34 : 1) was enriched in control samples and depleted in both the PEX and PEXG samples. Machine learning prediction with three supervised logistic regression binary classification tasks showed (1) POAG vs. control, with an 86% accuracy, (2) PEXG vs. control, with a 71% accuracy and (3) PEX vs. control, with an 86% accuracy. In conclusion, the analysis showed that the control (mean peak area 13.54 ± 6) had, on average, a higher total lipid content than the PEX, PEXG, and POAG AH samples. Elevations in Apolipoprotein A-I (APOA1) correlated with an increased abundance of PC lipid species in the AH of patients with PEXG. PC (18 : 0/18 : 2), PC (36 : 2), and PC (34 : 1e) are in low concentrations for PEX but are highly concentrated in PEXG, despite both having similar material deposits, suggesting that they are fundamentally different in composition.