Negative-ion hydrophilic liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) method has been optimized for the quantitative analysis of ganglioside (GM3) and other polar lipid classes, such as sulfohexosylceramides (SulfoHexCer), sulfodihexosylceramides (SulfoHex2Cer), phosphatidylglycerols (PG), phosphatidylinositols (PI), lysophosphatidylinositols (LPI), and phosphatidylserines (PS). The method is fully validated for the quantitation of the studied lipids in kidney normal and tumor tissues of renal cell carcinoma (RCC) patients based on the lipid class separation and the coelution of lipid class internal standard with the species from the same lipid class. The raw data are semi-automatically processed using our software LipidQuant and statistically evaluated using multivariate data analysis (MDA) methods, which allows the complete differentiation of both groups with 100% specificity and sensitivity. In total, 21 GM3, 28 SulfoHexCer, 26 SulfoHex2Cer, 10 PG, 19 PI, 4 LPI, and 7 PS are determined in the aqueous phase of lipidomic extracts from kidney tumor tissue samples and surrounding normal tissue samples of 20 RCC patients. S-plots allow the identification of most upregulated (PI 40:5, PI 40:4, GM3 34:1, and GM3 42:2) and most downregulated (PI 32:0, PI 34:0, PS 36:4, and LPI 16:0) lipids, which are primarily responsible for the differentiation of tumor and normal groups. Another confirmation of most dysregulated lipids is performed by the calculation of fold changes together with T and p values to highlight their statistical significance. The comparison of HILIC/ESI-MS data and matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) data confirms that lipid dysregulation patterns are similar for both methods. Graphical abstract ᅟ.

Department of Oncology Faculty of Medicine and Dentistry Palacký University and University Hospital 1 P Pavlova 6 775 20 Olomouc Czech Republic

Department of Urology Faculty of Medicine and Dentistry Palacký University and University Hospital 1 P Pavlova 6 775 20 Olomouc Czech Republic

Faculty of Chemical Technology Department of Analytical Chemistry University of Pardubice Studentská 573 532 10 Pardubice Czech Republic

Faculty of Pharmacy Hradec Králové Department of Organic and Bioorganic Chemistry Charles University Akademika Heyrovského 1203 500 05 Hradec Králové Czech Republic

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