## 6 Farber disease
### MALDI Non-Imaging MS Analysis of GSLs in Various Cell Cultures
#### 3.6.4 GSLs in Other Cell Lines  
In the study of PD, Lobasso et al. [286], profiling of lipids from human skin fibroblasts was performed, comparing samples from patients with PD (with Parkin protein present in the skin fibroblasts) with normal control samples. The analysis was performed on a Bruker Microflex LRF mass spectrometer (Bruker Daltonics, Bremen, Germany), and MALDI TOF/TOF Ultraflex Extreme mass spectrometer, with 9-AA (aminoacridine hemihydrate) used as a matrix, in both ion modes, positive and negative. Lipids extracted from the cell culture lines were dried under nitrogen gas and dissolved in chloroform for sample preparation, then underwent further analysis using TLC on silica gel plates and finally, the lipid band was extracted from the silica plates via chloroform/methanol, diluted with propanol/acetonitrile, mixed with the 9-AA matrix, and then analyzed via MS.  
GSLs in the range of GM3 (\(m/z\) 1151.7 d18:1/C16:0; \(m/z\) 1235.8 d18:1/C22:0; and \(m/z\) 1263.8 d18:1/C24:0) and GM2 (\(m/z\) 1354.8 d18:1/C16:0; \(m/z\) 1438.9 d18:1/C22:0; and \(m/z\) 1466.91 d18:1/C24:0) were identified in the parkin-mutant fibroblasts in negative ion mode, but were absent or present in undetectable amounts in the control samples, with significantly higher peaks in the patient samples for the gangliosides GM2 (C24:0) and GM3 (24:0). The results were replicated using intact fibroblasts (from a different patient) compared with commercial NHDF fibroblasts, with \(m/z\) 1466.9 (GM2, d18:1/C24:0) and \(m/z\) 1263.8 (GM3, d18:1/C24:0) being higher in patient samples, with an additional peak at \(m/z\) 1152.7 (GM3, d18:1/C16:0) found to be higher in patient samples. Specifically, the higher amounts of GM2 and GM3 in the parkin-mutant fibroblasts of PD patients compared to the control samples might be associated with the pathogenesis of PD, and these GSL markers could potentially aid in the early diagnosis of PD. Moreover, this study reveals a new approach based on MALDI TOF MS for studying PD using skin fibroblast samples as an alternative to brain tissue samples, offering a more accessible and convenient method of diagnosis. However, it is worth noting that the method has so far been applied only to cultured cell samples, and further research is needed to validate these findings in native tissue samples and to better understand the underlying mechanisms linking altered GSL profiles to PD development. In a study performed on Jurkat cells, Bond et al. [287], researched if mammalian cells are capable of synthesizing novel ganglioside products. For this purpose, metabolic oligosaccharide engineering was applied by inducing chemical modifications in various monosaccharides (including sialylation). Briefly, Jurkat cells were cultured with a diarizine-containing \(N\)-acetylmannosamine analog (AC\({}_{4}\)ManNDAz), with AC\({}_{4}\)GlcNAcNDAz (as a control line, these cells are not expected to display SiaDAz), and with Ac\({}_{4}\)ManNAc (to determine if exogenous addition of a normal metabolite in the sialic acid biosynthesis pathway affects ganglioside production). After extraction and purification, gangliosides were analyzed using HPTLC, and afterward using MALDI-TOF-MS on a Bruker Microflex instrument with THAP used as matrix, in negative ion mode. The obtained results showed that untreated Jurkat cells express several ganglioside species, with the naturally occurring form of sialic acid: monosialylated GM3, GM2, and GM1 species, having various ceramide configurations. The cell line culture that was treated with Ac\({}_{4}\)ManNDAz revealed that alongside the naturally occurring ganglioside species, several gangliosides containing SiaDAz were also present (SiaDAz-containing gangliosides that have lost N\({}_{2}\), in some cases), thus confirming the novel production of gangliosides.