## 6 Farber disease
#### 3.5.2 GSLs in Cancer
MS analysis was performed using DE-MALDI-TOF MS (Voyager Elite XL, Biospectrometry, PerSeptive Biosystems, Framingham, MA, USA), 2.5 DHB matrix, using positive ion mode), and the major ion peaks at \(m/z\) 871.78 and \(m/z\) 899/79 were identified as lysoGM3 (d18:1), \(\left[\mathrm{M_{1}-H}\right]^{-}-\mathrm{CH_{3}CO}\), and lysoGM3 (d20:1) \(\left[\mathrm{M_{2}-H}\right]^{-}-\mathrm{CH_{3}CO}\). The GM3 long-chain base composition was calculated as follows: d18:1 (77.4%), d18:0 (1.9%), d20:1 (20.5%), and d20:0 (0.2%). Serb et al. [216] analyzed a brain tumor sample (secondary brain tumor, originating in a non-small-cell lung carcinoma (NSCLC)) using MALDI-TOF MS, using a Bruker ultrafelXtreme apparatus and 2.5 DHB as a matrix, in negative ion mode. To separate the raw mixture of acidic GSLs from the brain tumor tissue, sequential extraction and purification procedures were performed [216]. TLC was used for separation control. The solvent was eliminated using nitrogen entrainment, and the resulting raw GSL mixture was dried in a desicator with the aid of P\({}_{2}\)O\({}_{5}\). A methanolic solution was used to dilute the native GSL mixture, to be further analyzed using MS. The GSL composition was characterized by the presence of glucccerebrosides and galactocerebrosides (at \(m/z\) 714.123, \(m/z\) 730.094, \(m/z\) 744.093, \(m/z\) 766.078, \(m/z\) 792.620, \(m/z\) 806.184, \(m/z\) 864.709, \(m/z\) 953.470), LacCer (at \(m/z\) 864.709, \(m/z\) 886.630, \(m/z\) 914.250, and \(m/z\) 974.073), and other hexosylCer (at \(m/z\) 1631.857--which were attributed to type I A antigen or/and type II A antigen). Additionally, asialylated gangliosides (GA2-type found at \(m/z\) 1007.031, \(m/z\) 1077.730, and \(m/z\) 1108.151) and sulfatides (found at \(m/z\) 766.078 and \(m/z\) 974.073) were detected. Neutral GSLs belonging to the _lacto-_, _neolacto-_, _globo-_, and _isoglobo-_ series were identified at \(m/z\) 1591.939 and \(m/z\) 1631.857. The gangliosides present included GM4, GM3, GM2, and GM1 (monosialylated), GD1, GD2, and GD3 (disialylated), and GT3 (trisialylated) structures, of which GD2 and GD3 are linked to metastasis. Unusual KDN and Neu5 Gc acids were observed to be present in GSL species KDN-GM1 (t16:1/12:0) and GalNAc \(\beta\) 1-4 (NeuGc \(\alpha\) 2-3) Gal \(\beta\) 1-4 Glc \(\beta\)-Cer (d18:1/20:0), respectively. These structures have been detected as \(\left[\mathrm{M-H}\right]^{-}\) ions in the MS1 spectrum at \(m/z\) 1407.710 and \(m/z\) 1426.274, respectively. Additionally, KDN was identified in another GSL species, namely, KDN \(\alpha\) 2-3 Gal \(\beta\) 1-4 (Fuc \(\alpha\) 1-3) GlcNAc \(\beta\) 1-3 Gal \(\beta\) 1-4 Glc \(\beta\)-Cer (d18:1/26:1), observed at \(m/z\) 1759.163. The GSL species detected at \(m/z\) 2286.826 as a minor peak has been identified as carbohydrate ligands of selectins, specifically sLea-x and sLea-Lex, with the following structure: Hex(4)-HexNAc(2)-Fuc(2)-NeuAc-Cer 42:1, O2. These specific antigens are usually not present in noncancerous tissue. In addition to the monosialyl Lewis antigens, another GSL exhibiting properties of a tumor-associated antigen, disalyl Lea, was identified at \(m/z\) 2092.106. To investigate the structure of the unusual GSL species carrying the KDN residue, the precursor ion [M \(-\) H\({}^{+}\)]- at \(m/z\) 1407.710, was further subjected to tandem MS in \"LIFT\" mode and the obtained fragmentation ions have fully documented the presence of the KDN-GM species with Cer (t16:1/12:0): the ion observed at \(m/z\) 1363.722 is a consequence of the decarboxylation of KDN, which is linked to the inner Gal residue; the presence of the potassiated KDN residue is confirmed by the ion at \(m/z\) 249.061, referred to as B1\(\beta\). Additionally, the ions at \(m/z\) 1227.648 and \(m/z\) 1245.659, labeled as \(Z3\alpha\) and Y3\(\alpha\), respectively, correspond to the KDN-GM1 (t16:1/12:0) species after the elimination of the external Gal residue.