## 3 MALDI Non-Imaging MS Analysis of GSLs
### The Influence of Matrices Used in MALDI Non-Imaging MS Analysis of GSLs
Distinguishing between GD1a and GD1b was challenging due to their structural isomerism (differing only in the position of a single sialic acid) [138]. Employing DHA as a matrix resulted in mass spectra where ganglioside molecular ions became the predominant peaks, accompanied by reduced fragment peaks [139]. A combination of DHA, ammonium sulfate, and heptafluorobutyric acid (HFBA) effectively curtailed salt adduct formation, enhancing signal detection due to the presence of ammonium sulfate. Simultaneously, HFBA bolstered DHA stability within a vacuum [125]. When using the negative linear ion mode, employing the DHA matrix enhanced the identification of ganglioside species in adult wild-type C57 BL/6 mice. This included O-acetylated GT1 forms, and a broader range of sialylated gangliosides, notably GD1 and GQ1 and their acetylated variants. These improvements were accompanied by a decrease in the S/N and an increase in resolution [125]. * **CHCA (\(\alpha\)-cyano-4-hydroxycinnamic acid)** This matrix is usually used for lower-mass-range peptides, and it is well soluble in organic solvents and insoluble in water. The analyte molecules receive a lot of internal energy during desorption and ionization, which labels this as a \"hard\" matrix. A considerable amount of ion fragmentation appears in the drift tube (post-source decay). As the analyte molecules become bigger, the probability of fragmentation increases until fragmentation of almost all of the analyte ions occurs. Consequently, CHCA is the choice matrix for the analysis of post-source decay (PSD). The main advantage of CHCA is the ability of this matrix to form small homogeneous crystals. Geometric inhomogeneity is related directly to decreased resolution in the MALDI analysis, therefore, CHCA usage commonly produces good resolution. As CHCA is not water soluble, the samples can be washed on the target. Fragmentation occurs as well as in the case of DHB in positive ion mode. Typical acidic GSLs (GM1, GM2, GD2, GD1a, GD1b, and GT1a) were characterized by E. Ito et al. with an AP-MALDI-QIT-TOF MS with MS\({}^{n}\) switching using CHCA as the matrix. They analyzed multiple gangliosides from different tissues: GM1 was derived from bovine brain, while GM2 was sourced from the brain of a patient with Tay-Sachs disease. GD2, GD1a, and GD1b were obtained from bovine brain, and GT1a from a human brain. Both positive and negative ion modes were employed, yielding data that proved valuable for the structural analysis of the compounds. MS1, MS2, and MS3 experiments were conducted in the positive ion mode. The mass spectra revealed sodium adduct molecular ions of GM1 containing Cer (d18:1/18:0) and (d20:1/18:0) at \(m/z\) 1568 and 1596, respectively. Ions resulting from the removal of Neu\(\Delta\)c from the sodium adduct molecular ions were observed at \(m/z\) 1277 and 1305 in the MS spectrum. In MS2 spectra, ions at \(m/z\) 1115 and 912 were attributed to the elimination of Hex and HexNAc, while ions at \(m/z\) 1143 and 940 were linked to the same eliminations. As ceramide cleavage was produced during MS2, it became impossible to perform structural characterization of the ceramide moieties of GM1 by selecting \(m/z\) 912 and 940 as the precursor ions [140]. For characterization of GM2, CHCA seemed a suitable matrix as well as for FucGM1, as no fragmentation of fucose was observed [124], although DHB was superior to CHCA, minimizing carbon dioxide and sialic acid loss from GSLs. Ample fragmentation is produced with the absence of both the intact disialoganglioside and the major monosialoganglioside fragments [121]. The CHCA matrix proved inadequate, resulting in a low S/N and significant sialic acid decay. Consequently, trisialogangliosides (GT1) were entirely undetectable as molecular ions [117]. The spectra displayed extensive sialic acid loss, manifesting as broad, unresolved peaks. The obtained fragment ions were broad and poorly defined; however, some structural insight could still be derived.