## 3 MALDI Non-Imaging MS Analysis of GSLs
### MALDI Non-Imaging MS Analysis of GSLs in the Healthy State
#### 3.4.2. GSLs from Human Brain Tissues
For GD1a and GalNAc-GD1a, methyl esterification with methyl iodide was necessary to minimize sialic acid loss and obtain good MS spectra, whereas for the \"b\" series gangliosides (GD1b, GD2), there was no need for methyl esterification, as they are evidenced in the mass spectra as dehydrated molecule-related ions \(\left[\mathrm{M}-\mathrm{H}\right]^{-}-\mathrm{H}_{2}\mathrm{O}\) at \(m/z\) 1817.97 and \(m/z\) 1846.00, suggesting a lactone ring formed in the disialic acid moiety, between the hydroxygroup and the carboxyl group. Using this method, the authors were able to differentiate between the same-molecular-weight GD1a and GD1. GD1a exhibited methyl esterification of its sialic acid residues to prevent their loss during the positive ion mode analysis. This modification was indicated by high peaks at \(m/z\) 1887.95 and \(m/z\) 1915.98 corresponding to [M + Na]\({}^{+}\) monoisotopic masses for the dimethyl ester of GD1a. In contrast, GD1b lacked such methyl esterification, resulting in well represented peaks at \(m/z\) 1858.68 and \(m/z\) 1886.79 corresponding to [M + Na \(-\) 2H]\({}^{-}\) monoisotopic masses. Consequently, the differential modification of sialic acid residues in GD1a and GD1b led to distinct mass spectra and discernible peaks, thereby enabling their differentiation. A remarkable observation was that various series of gangliosides have individually characteristic molecule-related ions peaks (for \"a\" series [M + Na \(-\) 2H]\({}^{+}\) and for \"b\" series [M - H]\({}^{-}-\) H\({}_{2}\)O). In a study by Gizaw et al. [159], normal and pathological (Alzheimer's disease) human brain tissues (collected from the parietal, temporal, frontal, and occipital cortices), and blood serum are used to compare glycans using MALDI-TOF and, afterward, MALDI-TOF/TOF mass spectrometry on a Bruker Daltonics apparatus (Bruker Daltonics, Bremen, Germany), using DHB as a matrix. A novel method of glycolotting was proposed by the authors, which enables a process of efficient glycan enrichment that purifies and labels GSL-released glycans. Total lipids were extracted from the brain tissue samples through CHCl\({}_{3}\)/MeOH/H\({}_{2}\)O. The upper phase was subsequently dried at room temperature, suspended in CHCl\({}_{3}\)/MeOH, sonicated, and subjected to ozonolysis and alkaline degradation to break down the GSLs, followed by glycolotting using BlotGlyco beads for glycan enrichment. Methyl esterification and BOA labeling were performed to further modify the glycans. This method allows for effective analysis of the labeled glycans using MALDI-TOF-MS [134]. It was found that the intact oligosaccharides release of glycan, independently of GSLs' structures, was obtained indiscriminately by the ozone-based protocol proposed. The study found abundant gangliosides in both diseased and normal samples. The mass spectrum revealed the highest intensity peak of the ganglioside GM1, followed by GM2, GD1a/GD1b-lactone and GT1a/GT1b-lactone. It should be mentioned that the GD1b-lactone ganglioside is not an artifact, but rather an authentic product of brain metabolism which is human brain specific [134]. Taki T. [158] introduces a novel approach known as TLC-Blot-MALDI-TOF MS, where the separation of lipids on a TLC plate is followed by marking them with a soft pencil after being treated with the primuline reagent to enhance their visibility under UV light. The plate is subsequently immersed in a blotting solvent for 10 s, and immediately placed onto a PVDF membrane (glass fiber filter/Teflon membrane/PVDF membrane/HPTLC plate), which is then pressed in a thermal blotter for 30 s at a temperature of 130 \({}^{\circ}\)C. The lipids are transferred to the opposite side of the membrane, facing away from the TLC plate. Distinct bands corresponding to GM1, GD1a, GD1b, and GT1b lipids were clearly visible on the TLC plate when analyzing lipids extracted from brain samples of individuals with Alzheimer's disease, Parkinson's disease, and from normal human brain control samples.