## 3 MALDI Non-Imaging MS Analysis of GSLs
### MALDI Non-Imaging MS Analysis of GSLs in Pathology
#### 3.5.1 GSLs in Storage Diseases
Folch's partitioning with chloroform/methanol/water was utilized and the lower phase underwent mild alkali treatment to decompose glycosphingolipids, followed by acidification and extraction with hexane to obtain fatty acids and cholesterol in the upper phase and crude alkali-stable sphingolipids in the lower phase. Sugiyama et al. [123] approached several storage disorders, with tissue samples from the liver and spleen of different autopsied patients using CHCA and DHB matrices on a DE-MALDI-TOF MS (Voyager Elite XL, Framingham, USA), in both positive and negative ion mode. The preparation of samples was made using Folch's partitioning as previously described. Using splenic tissue as a sample from a patient with GM1 gangliosidosis, the mass spectra obtained using MALDI-TOF MS revealed peaks at \(m/z\) 1517 and 1545, which are consistent with the accumulation of GM1 in the tissue sample (GM1 (d18:1C16) \(\left[\mathrm{M-H}\right]^{-}\) and GM1 (d18:1C18) \(\left[\mathrm{M-H}\right]^{-}\), respectively). Collectively, these investigations underscore the critical role played by the accumulation of GM1 ganglioside in driving the pathogenesis of GM1 gangliosidosis. The perturbed buildup of GM1 ganglioside disrupts normal cellular processes, leading to the symptomatic manifestation and functional impairment observed in affected individuals. Applying techniques such as MALDI MS empowers researchers to scrutinize and quantify GM1 ganglioside levels in tissues, thereby unraveling the intricate molecular foundations of GM1 gangliosidosis and related storage disorders. 2. Tay-Sachs disease  
Tay-Sachs disease is primarily characterized by the accumulation of GM2 ganglioside in the cells, particularly nerve cells, due to a deficiency in the enzyme hexosaminidase A (Hex-A). This deficiency leads to the inability to break down GM2 ganglioside, resulting in its accumulation within lysosomes, the cell's waste disposal organelles. As a result, the affected cells become engorged with these lipid molecules, leading to cellular dysfunction and eventually causing the symptoms associated with Tay-Sachs disease [186]. A sample of brain tissue obtained from an individual diagnosed with Tay-Sachs disease was employed for determining GM2 levels by Sugiyama et al. [151]. GM2 was found at \(m/z\) 1382.8 and 1410.8 (monoisotopic masses \(\left[\mathrm{M-H}\right]^{-}\) d18:1-C18:0 and d20:1-C18:0, respectively), with no sialic acid loss (both 2.5 DHB and \(\alpha\)-CHCA were used as matrices, with good results). Ito et al. [140] used a brain tissue sample from a Tay-Sachs diseased patient with an emphasis on the long-chain base of GM2 using a QIT (quadrupole ion trap)-TOF MS (Shimadzu, Japan), together with an AP (atmospheric pressure)-MALDI source (Mass Technologies, MD); nitrogen laser at 337 nm wavelength and Ar gas pulsed for ion cooling and CID. CHCA was used as the matrix. A comprehensive protocol is described by the authors [123]. For identifying the long-chain base composition of GM2, the authors used MS, MS\({}^{2}\), and MS\({}^{3}\) of \(m/z\) 1382 and 1410 \(\left(\left[\mathrm{M-H}\right]^{-}\right)\), as identified in the sample. The precursor ion was selected at \(m/z\) 1382, and then the MS\({}^{2}\) spectra was performed, the ceramide ion at \(m/z\) 564 (\(\left[\mathrm{M-H-NeuAc-HexNAc-Hex}\right]^{-}\)) was set aside for further analysis through MS\({}^{3}\), obtaining peaks at \(m/z\) 324 (S), 308 (T), 282 (U), 263 (R), and 237 (P), which represents the ceramide with a C18:0 fatty acid and d18:1 sphingosine. Similarly, following a comparable approach, the precursor ion with \(m/z\) 1410 was chosen, and \(m/z\) 592 was selected from the MS\({}^{2}\) spectra to undergo additional fragmentation. This resulted in significant peaks observed at \(m/z\) 324 (S), 308 (T), 291 (R), 282 (U), and 265 (P/V), representing the ceramide structure with d20:1 sphingosine and C18:0 fatty acid [125; 140].