## 6 Farber disease
### MALDI Non-Imaging MS Analysis of GSLs in Various Cell Cultures
#### 3.6.4 GSLs in Other Cell Lines
The novel species were identified as follows: GM2--SiaDAz (at \(m/z\) 1425.6 and \(m/z\) 1530.0), GM2-SiaDAz--N\({}_{2}\) (at \(m/z\) 1397.5 and \(m/z\) 1507.9), GM1-SiaDAz (at \(m/z\) 1580.0 and \(m/z\) 1698.4), GD1a--NeuAc/SiaDAz at \(m/z\) 1879.8 and at \(m/z\) 1902.7, GD1a-SiaDAz/SiaDAz (at \(m/z\) 1948.1, \(m/z\) 1971.0 and \(m/z\) 1991.1), and GD1a-SiaDAz/SiaDAz at \(m/z\) 2059.3. This research holds several biological significances: (1) the diversity of ganglioside structures--the study demonstrates that Jurkat cells can synthesize gangliosides with modified structures, while continuing to produce their natural gangliosides, expanding the diversity of ganglioside species that can be present on cell surfaces, having implications for cell signaling, immune response regulation, and other cellular processes; (2) cell-to-cell interactions--gangliosides are involved in cell-cell interactions, particularly in immune responses, and the ability of Jurkat cells to generate novel ganglioside products suggests that cells could modulate their interactions with other cells in response to specific environmental signals or during immune responses; (3) metabolic engineering tools--the study showcases the utility of metabolic oligosaccharides engineering as a tool for probing the glycosylation pathways of cells. This approach enables researchers to investigate the roles of specific glycan structures in cellular functions; and (4) characterizing ganglioside-mediated interactions--by generating novel gangliosides, researchers can explore how these modified structures might influence cell adhesion, signaling, and immune responses. This information could have implications for understanding diseases where altered ganglioside expression or structure is implicated. Liang et al. [288] employed MALDI MS profiling (on an ABI 4700 Proteomics Analyzer, Foster City, CA, USA, using 2,5 DHB as a matrix) and MS/MS sequencing (low- and high-energy CID MALDI MS/MS performed on a Q/TOF Ultima MALDI with ACHC as a matrix, as well as the 4700 Proteomics Analyzer with 2.5 DHB as a matrix, in positive ion mode), to investigate the profiles of GSLs in human embryonic stem cells (hESCs) and differentiated cells derived from embryoid bodies (EBs). The cells were subjected to Folch's partitioning, obtaining total crude GSL extracts, which were further permethylated (to neutralize the carboxylic groups on sialic acids through methyl esterification, thereby enabling the characterization of neutral and negatively charged sialylated species). MALDI MS and MS/MS analysis were performed without further purification of the samples. The results revealed distinct GSL species present in these cell types. Undifferentiated human embryonic stem cells (hESCs) exhibit distinct glycosphin-golipids (GSLs) associated with the globo-series. Notably, Gb5 Cer (also known as SSEA-3), sially Gb5 Cer (SSEA-4), and fucosyl Gb5 Cer (Globo H, which carries a terminal Fuc-Gal--\"H antigen\") serve as prominent markers. These GSLs are linked to epitopes relevant to pluripotency and the undifferentiated state of hESCs. In contrast, differentiated EB cells suppress globo-series GSLs and predominantly display _ganglio_-series GSLs, the most prominent being GM3 and GD3, with the latter being highly expressed specifically in EB cells. Other ganglio-series GSLs, including GM2 (at \(m/z\) 1617.1), GM1a/GM1b (at \(m/z\) 1933.3), and GT1a/GT1b/GT1c (at \(m/z\) 2654.7), were also detected in lower abundance in EB cells. The ceramide moieties of _ganglio_-series GSLs in EB cells exhibited greater heterogeneity compared to those in hESCs, with variations in fatty acyl chain lengths. Theircomposition included mainly C16:0 and C18:0 fatty acids (FAs), with the exception that larger sialylated GSLs exhibited extended FA chains reaching up to C24:0/C24:1. Further analysis identified isomers of GM1a/GM1b and GD1a/GD1b in EB cells, indicating the presence of structural variants within the _ganglio_-series GSLs. The FA contents of these GSLs varied, with different species exhibiting distinct compositions.