## 3 MALDI Non-Imaging MS Analysis of GSLs
### The Influence of Matrices Used in MALDI Non-Imaging MS Analysis of GSLs
Discrimination between GD1a (with sialic acids on different sugars) and GD1b (with sialic acids linked together)was achieved based on the relative abundance of ions resulting from the elimination of one or two sialic acid residues. Notably, GD1b exhibited a more pronounced loss of two sialic acids [114]. Methyl esterification reduces fragmentation, as seen in the observations of Andrew K. Powell in a study conducted on GD1a (two sialic acid (Neu-5-Ac) residues a2-3-linked to Gal). When derivatized as the methyl ester, however, an abundant molecular ion was produced and weak fragmentation was observed, obtaining structural information on the oligosaccharidescharide chain. In the positive ion mode, this does not cause complete stabilization, as abundant fragment ions are present still [141]. Details regarding the carbohydrate sequences of asialoganglioside structures were acquired through AP-MALDI-QIT-TOF MS, utilizing MSn switching every 1.9 s per cycle in the negative ion mode. MS2 spectra of monosiologanglioside ions were generated for various gangliosides: GM1 at \(m/z\) 1544 and 1572, GM2 at \(m/z\) 1382 and 1410, GD2 at \(m/z\) 1382 and 1410, and GT1a at \(m/z\) 1544 and 1572. MS3 spectra of gangliosides, using ceramide ions as precursors, revealed distinctive types of ions (P, S, T, U, V, and R) associated with ceramide fragmentation, except for GD2. Despite the absence of the S-ion at \(m/z\) 324, ceramide structures of GD2 were still effectively characterized [125; 140]. The analysis of various lysogangliosides in the negative ion mode proved superior to the positive ion mode, primarily due to the suppression of alkali metal adduct formation and salt complexation. This also highlighted the superiority of CHCA as a matrix compared to DHB [128]. CHCA was employed as a matrix in several studies with the aim of structural characterization of GSLs related to different diseases based on MALDI MS, [137; 142]. Peaks corresponding to GM1 gangliosides (\(m/z\) 1517 and 1545) and asialo GM1 gangliosides (\(m/z\) 1277 and 1305) were detected in tissue samples from a patient with GM1 gangliosisosis [137]. Conversely, patients with other conditions exhibited a normal composition of long-chain bases in GM1 [142]. The sphingoid base of GM1 in Spielmeyer-Vogt type juvenile amaurotic idiocy seemed abnormal--a patient with Gaucher disease had a long-chain base composition in GM1 similar to the composition observed in the patient with Spielmeyer-Vogt idiocy [142]. * **5-methoxysalicylic acid (MSA)**  
Lee et al. [129] analyzed brain ganglioside GD1a extracted from porcine brain. They achieved high-quality ganglioside profiles in the positive ion mode while minimizing the loss of sialic acid residues. While DHB enabled the detection of intact GD1a ions, significant loss of sialic acid residues was observed, and the spectrum was predominantly composed of asialoganglioside ions resulting from the removal of two sialic acid residues. In contrast, differences in sialic acid loss between MSA and DHB in the negative ion mode were not as pronounced as in the positive ion mode. Unfocused post-source decay (PSD) fragment ions of GD1a were present in the range of \(m/z\) 1500-1600 in the positive ion mode, but these PSD fragment ions were effectively mitigated by reducing the relative concentration of gangliosides to MSA [129]. In another study, Park et al. [143] employed alkali earth metal additives in order to differentiate ganglioside isomers through tandem MS. Their analysis focused on gangliosides GD1a and GD1b extracted from bovine brains. While GD1a and GD1b could not be discerned in the first-order mass spectra in the positive ion mode, they generated metastable fragment ions with notably distinct characteristics. However, MALDI-TOF/TOF mass spectra facilitated the detection of specific fragment ions for both GD1a and GD1b isomers.