## 6 Farber disease
### MALDI Non-Imaging MS Analysis of GSLs in Various Cell Cultures
#### 3.6.1 GSLs in Breast Cancer Cell Lines
[269] determined the GSL composition of three breast cancer cell lines: Hs578T, MCF-7, and MDA-MB-231. Prior to MS analysis, after multiple steps of extraction and purification [44], the separated GSLs were permeabilized by incubating them for 2 h in a suspension that contained sodium hydroxide, dimethyl sulfoxide, and methyl iodide, extracted in methanol, and then washed with water. After the reagents evaporated, the samples were diluted in chloroform and applied to a 2.5 DHB matrix. For the MCF-7 cell line, the main ganglioside expressed was GM1a, confirmed by MS/MS (at \(m/z\) 1933.2, with Cer (d18:1/24:0) and at \(m/z\) 1821.1 with Cer (d18:1/16:0), followed by GM2 (at \(m/z\) 1729.1 with Cer (d18:1/24:0) and \(m/z\) 1617.0 with Cer (d18:1/16:0)), and GM3 (at \(m/z\) 1484.0 with Cer (d18:1/C24:0) and \(m/z\) 1371.9 with Cer (d18:1/16:0)). In addition, there were two peaks at \(m/z\) 2182.3 with Cer (d18:1/16:0) and \(m/z\) 2294.4 with Cer (d18:1/24:0), identified as GD1a and GD1b mixtures (in a proportion of 90% GD1a and 10% GD1b). _Globo_-series Gb4 was also identified at \(m/z\) 1572.0 (d18:1/C24:0) and at \(m/z\) 1459.9, having Cer (d18:1/16:0). The HS578T cell line expressed GM2 as the main ganglioside, according to the ion abundance observed in the mass spectra (at \(m/z\) 1729.1 with Cer (d18:1/24:0) and \(m/z\) 1617.0 with Cer (d18:1/16:0)), followed by GM1b (at \(m/z\) 1933.2 with Cer (d18:1/24:0))--confirmed by MS/MS fragmentation. GM3 was also identified at \(m/z\) 1371.9, having Cer (d18:1/C16:0) and \(m/z\) 1484.0 with Cer (d18:1/24:0) but in lower abundance, and GD2 was identified at \(m/z\) 2090.3 and Cer (d18:1/24:0). A low amount of GA2 was detected at \(m/z\) 1367.9, having Cer (d18:1/24:0) and \(m/z\) 1255.8 with Cer (d18:1/16:0), and Gb4 was present in a trace amount, almost absent. The MDA-MB-231 cell line expressed a large variety of GSLs, the main ganglioside detected being GM1b, confirmed by MS/MS fragmentation (at \(m/z\) 1933.3 with Cer (d18:1/24:0) and at \(m/z\) 1821.1, Cer (d18:1/16:0)), followed by GM3, (at \(m/z\) 1484.0, Cer (d18:1/24:0) and \(m/z\) 1371.9 Cer (d18:1/16:0)), and GM2 (at \(m/z\) 1729.1, Cer (d18:1/24:0)). A mixture of GD1a (40%) and GD1c (60%) was determined from the peaks corresponding to \(m/z\) 2182.3, Cer (d18:1/16:0) and \(m/z\) 2294.4, Cer (d18:1/16:0). _Globo_-series GSLs were also detected at \(m/z\) 1460.0 (Gb4, and Cer (d18:1/16:0)), \(m/z\) 1572.1 (Gb4, and Cer (d18:1/24:0)), \(m/z\) 1326.9 (Gb3, and Cer (d18:1/24:0)), and \(m/z\) 1214.8 (Gb3, and Cer (d18:1/16:0)), as well as LacCer at \(m/z\) 1010.75 and \(m/z\) 1122.88. The studies performed with MALDI MS on breast cancer cell cultures indicate that there is a large variety of GSLs expressed by the cell lines, but also the fact that the GSLs expressed are highly specific to a certain type of cell. All investigated wild-type breast cancer cell lines expressed a-series gangliosides GM1, GM2, and GM3 (except for SUM159 PT, MCF-7, and MDA-MB-468), while b-series gangliosides were limited to Hs587T, SUM159 PT, and MDA-MB-231 in low amounts (GD2) and to MDA-MB-468 (GD3), and c-series gangliosides were absent. O-acetylated gangliosides (OAc-GD3 and OAc-GD2) were present only in Hs587T and SUM159 PT cell lines. Overexpression of GD3 synthase showed to increase the expression of _b-series_ gangliosides (GD2 in MCF-7 GD3S+ and in MDA-MB-231 GD3S+, while GD3 in MDA-MB-231 GD3S+, MDA-MB-468 GD3S+ and MCF-7 GD3S+), of _c-series_ gangliosides (GT2 and GT3 in MDA-MB-231 GD3S+ and MDA-MB-468 GD3S+), and of O-acetylated gangliosides(OAc-GD3, OAc-GD2, OAc-GT3, and OAc-GT2 in MDA-MB-231 GD3S+, together with OAc-GD2 and OAc-GT3 in MCF-7 GD3S+). _B-series_ gangliosides GD3 and GD2 have been described as tumor-associated carbohydrate antigens (TACA) in neuroectoderm-derived tumors, as well as in breast cancer [270].