## 3 MALDI Non-Imaging MS Analysis of GSLs
### The Influence of Matrices Used in MALDI Non-Imaging MS Analysis of GSLs
Double sodium adduct ions were also found, which shifted the mass by + 22 Da compared to the [M + Na]\({}^{+}\) ions. Other peaks were identified in the range between 912 and 997 Da with the same 28 Da intervals as above. The mass shift of 291 Da is attributed to the loss of a labile sialic acid [118]. Sodium and potassium ion adducts, especially for GD1, and GM3 gangliosides corresponding to the sodium-coordinated species such as [M - H + 2 Na]\({}^{+}\), [M + Na]\({}^{+}\), and [M + Na]\({}^{+}\)\(-\) CO\({}_{2}\), were observed by Colsch B et al. [125] in a study on brain GSLs from adult wild-type C57 BL/6 mice; NeuAc-dissociated ions and NeuAc-Hex-dissociated ions were also observed in a study by Kamimiya H. et al. using MALDI-TOF MS on GM3 standard GSL isolated from bovine brain and GM1a GSL isolated in their laboratory [132]. They also managed to reduce background noise by coupling high-performance thin-layer chromatography (HPTLC) with MALDI MS after washing the dried sample from a glass tube with non-polar organic compounds such as 1,2-dycholoroethane. This was performed to counter impurities from contaminants co-extracted with the samples, such as synthetic polymers, starch, and calcium sulfate, which are used as binders. The inclusion of spermine with DHB facilitated enhanced crystal formation and seemed to reduce the formation of alkali adducts, enabling sample analysis without the need for desalting, as observed by Meshref Y et al. [133] in a study on bovine brain GSLs using MALDI MS. In the negative ion mode, deprotonated ions are formed, for example, three separate types of molecular ions were observed, [M - H]\({}^{-}\), [M - 2H\({}^{+}\) + Na]\({}^{-}\), and [M - H]\({}^{-}\)\(-\) H\({}_{2}\)O. The smallest amount of fragmentation was produced in the negative ion mode (even though considerable fragmentation may occur in both negative and positive ion modes); some fragmentation was deemed acceptable because the fragmentation degree is usually consistent under identical conditions of ionization and can be accounted for within the analysis, as observed by Lee H et al. [122] in their study on human and bovine milk GSLs using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Taketomi T et al. [128] used MALDI MS on brain gangliosides obtained from different species (GM1, GM2, GM3 from human brain, rat brain GM1 and GD1a, porcine brain GM1) and observed that negative ion mode is preferred (both when using DHB as well as CHCA as a matrix) because it suppresses salt formation and the production of alkali metal adducts. Moreover, DHB was shown to be the matrix of choice in various studies analyzing GSLs profiles in a multitude of diseases. Alterations in the expression levels of GSLs provide potential serum biomarkers in disorders of the central nervous system and can also contribute to the pathogenesis of neurodegenerative diseases [134]. Thus, Gizaw et al. [134] showed that GM1, GM2, and GM3 seemed to be altered in the brain and serum samples of Alzheimer's disease patients, while Holst et al. [135] and Du et al. [136] observed aberrant glycosylation of GSLs in cancer of the colorectum and in hepatocarcinoma, respectively (a distinct upregulation of GM1 and GM2 in hepatocarcinoma; more detailed information is provided in Sections 3.5 and 3.6). Additionally, Fujiwaki et al. [137] showed the occurrence of peaks suggesting GM1 gangliosides (\(m/z\) 1517 and 1545) in negative ion mode and asialo GM1 gangliosides (\(m/z\) 1277 and 1305) in positive ion mode in all sample tissues (liver, spleen, cerebellum, cerebellum) of a GM1 gangliosides patient, when analyzed using MALDI-TOF-MS.  
* **DHA (2,6-dihydroxyacetophenone)** Jackson et al. [138] described the use of the DHA matrix in addition to DHB for direct MALDI-TOF MS lipid analysis of the rat cerebellum. GM1, GD1, and GT1 gangliosides assigned mass peaks were identified in the cerebellar cortex. Additionally, mass peaks linked to GM1 were identified in the cerebellar peduncle.