Full scan (500C1800, two microscans, maximum 100?ms, target value of 30,000) was performed, followed by data-dependent MS2 scans (two microscans, maximun 100?ms, target value of 10.000) with normalized collision energy of 35%, isolation window of 2.5 units, activation q?=?0.25 and activation time 30?ms). A determinant (C). The lanes were: lane 1, reference total non-acid glycosphingolipids of human blood group AB erythrocytes, 40?g: lane 2, reference calf brain gangliosides, 40?g: lane 3, total non-acid glycosphingolipids of GIST I, 40?g; lane 4, total non-acid glycosphingolipids of GIST II, 40?g. Characterization of total acid GIST glycosphingolipids LC-ESI/MS The native total acid glycosphingolipids fractions from GIST I and II were analyzed by LC-ESI/MS (exemplified in Fig.?3). In both cases the major molecular ions were from your GM3 ganglioside with d18:1C16:0 ceramide (1151) and d18:1C24:1 ceramide (1261). In the case of GIST II there were also molecular ions of the GD3 ganglioside with d18:1C24:1 ceramide (776) and the GD1a ganglioside with d18:1C24:1 ceramide (959). A minor ion at 794 was found by a search for molecular ions of sulfatide, and MS2 of this ion gave a characteristic sulfatide spectrum (Supplemental Physique S1)15. Open in a separate window Physique 3 Base peak chromatogram from LC-ESI/MS of the total acid glycosphingolipid portion from GIST II. The identification of glycosphingolipids was based on their retention occasions, determined molecular people and following MS2 sequencing. The info had been prepared using the Xcalibur software program (edition 2.0.7, Thermo Scientific, www.thermofisher.com). The glycosphingolipids determined in the chromatograms had been: Sulfatide, SO3-3Gal1Cer; Neu5Ac-GM3, Neu5Ac3Gal4Glc1Cer; Neu5Ac-GD3, Neu5Ac8Neu5Ac3Gal4Glc1Cer; Neu5Ac-GD1a, Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4Glc1Cer. In the shorthand TP-10 nomenclature for fatty bases and acids, the number prior to the colon identifies the carbon string length and the quantity after the digestive tract provides final number of dual bonds in the molecule. Essential fatty acids having a ATP1A1 2-hydroxy group are denoted from the prefix h prior to the abbreviation 1151 and 1263. MS2 of the ions determined the GM3 ganglioside with d18:1C16:0 and d18:1C24:0 ceramides, respectively (data not really shown). Several doubly billed ([M???2H+]2?) molecular ions had been acquired by LC-ESI/MS of small fraction A-2. MS2 determined the main molecular ion as the GD1a ganglioside with d18:1C16:0 ceramide (903), d18:1C22:0 ceramide (946), TP-10 and d18:1C24:0 ceramide (960) (Fig.?4C). The current presence of the GD1a ganglioside in small fraction A-2 was good binding of anti-GD1a antibodies to the small fraction (Fig.?4B, street 3). There were [M also???2H+]2? ions from the GD3 ganglioside (721, 763 and 777), and of Neu5Ac-neolactotetraosylceramide or the GM1 ganglioside (814). The bottom peak chromatogram from small fraction A-3 was weakened and only got one [M???2H+]2? ion at 777 related towards the GD3 ganglioside and one [M???2H+]2? ion at 960 related TP-10 towards the GD1a ganglioside, both with d18:1C24:0 ceramide (data not really demonstrated). [M???2H+]2? ions at 777 with 960 related towards the GD3 and GD1a gangliosides had been also discovered by LC-ESI/MS of small fraction A-4 (Fig.?4D). Right here the major substance offered a [M???2H+]2? ion at 1105.5. MS2 of 1105.5 proven a ganglioside with three Neu5Ac, three Hex and one HexNAc and d18:1C24:0 ceramide (Fig.?5A). The ion at 581 proven a Neu5Ac-Neu5Ac series, as the ions at 1920, 1629 and 1338 had been due to lack of one, two and three Neu5Ac through the molecular ion. MS3 of 960 also offered ions at 1629 and 1338 due TP-10 to lack of one and two Neu5Ac, and in addition an ion at m/z 972 because of lack of a Hex and a HexNAc (Fig.?5B). Used together this proven a GT1b ganglioside with d18:1C24:0 TP-10 ceramide (Fig.?5C). Very much the same the GT1b ganglioside with d18:1C16:0 ceramide was determined by MS2 and MS3 from the [M???2H+]2? ion at 1049.5, as well as the.