## 3 MALDI Non-Imaging MS Analysis of GSLs
### The Influence of Matrices Used in MALDI Non-Imaging MS Analysis of GSLs
However, compared to electrospray ionization (ESI), MALDI ionization has a better tolerance to impurities, because the desorption process helps free the analyte ions from the undesirable impurities in a similar way as it frees them from the matrix. Zarei et al. [117, 127] analyzed mouse brain gangliosides using MALDI-TOF using multiple matrices. Various solvents such as ethanol/water (1:1, \(v/v\)), acetonitrile/water (7:3, \(v/v\)), and ethanol/water/acetone (5:4:1, \(v/v\)) were used as solvents for different matrices DHB, ATT/DAC, and 2,4,6-trihydroxyacetophenone/DAC and these mixtures were examined initially using direct MALDI MS in positive ion mode. Efficient ionization of neutral GSL species was the target of this MS approach. The DHB matrix was found to provide a higher signal-to-noise ratio (S/N) compared with other matrices such as CHCA, even though a significant loss of sialic acid was observed. This occurred more in trisialo-GSLs compared to mono- or disialo-GSLs. Afterwards, DHB was dissolved in the aforementioned solvents and subjected to nano-high-performance liquid chromatography MALDI MS (nanoH-PLC/MALDI MS). Taking into consideration the crystallization efficiency without the need for any recrystallization attempts and the abundant yield coverage of major and minor neutral GSL species from the samples analyzed within spots, the DHB matrix dissolved in a mixture of acetonitrile/water (7:3, \(v/v\)) was best suited. One major drawback of MALDI MS is the formation of fragmentation ions [117, 118, 119, 120]. However, Penn S. et al. [121] found that DHB was a suitable matrix to limit fragmentation on a mixture of ganglioside standards when they compared MALDI with ESI in their study on Fourier transform mass spectrometry (FT-MS). Both positive and negative ion modes were employed, leading to fragmentation in both cases. Negative ion mode exhibited superior performance for GSL analysis. When using DHB as a matrix, MALDI spectra in negative ion mode outperformed those in positive ion mode. The lower performance in positive ion mode can be attributed to the heightened susceptibility of sialic acid to charge-induced fragmentation. Sugiyama et al. [123] analyzed different GSLs from different tissues (ganglioside GM1--isolated from GM1 gangliosides brain, GM2--from a Tay-Sachs brain, GM3 and GD3--from human brain tumor tissue) in both positive and negative ion modes and found that using DHB minimizes sialic acid, fucose, and carbon dioxide loss. More fragmentation was observed in positive ion mode, prompting methyl esterification (methylesterification neutralization of all sialic acids carboxylic groups) of the gangliosides at the carboxyl groups of the sialic acids to reduce the loss of sialic acid [123]. Mank M. et al. [124] used MALDI MS on the GM1 brain ganglioside and found that the addition of butylamine diminished the intensity of fragment ions during the analysis of labile monosialylated oligosaccharides and bovine ganglioside GM1. Negative ion mode was not extensively investigated, as preliminary tests involving DHB and butylamine indicated that signal intensities were approximately 3 to 15 times lower in negative ion mode compared to positive ion mode. Gangliosides in positive ion mode form alkali metal adducts ([M + K]\({}^{+}\) and [M + Na]\({}^{+}\)) [10, 125, 131, 132]. Bode et al. [118] used MALDI-TOF MS on GSLs obtained from bovine and human milk and obtained a [M + Na]\({}^{+}\) adduct of a 36 C atom ceramide-residue of GM3 ganglioside. The mass distribution designated to GM3 was in the range 1176 to 1326 Da, while the molecular masses of GD3 ranged between 1523 and 1624 Da. These mass peaks can be caused by MALDI-induced formation adduct ions, ganglioside-alkali ions such as [M + Na]\({}^{+}\) and [M + K]\({}^{+}\). There are 28 Da intervals within the peak cluster which can be associated to chain length variations in the sphingoid base or fatty acid with CH\({}_{2}\) groups of even number.