## 3 MALDI Non-Imaging MS Analysis of GSLs
### Instrumentation in MALDI Non-Imaging MS Analysis of GSLs
As a consequence, the signal intensities attainable, marked by fluctuations occurring from one laser shot to another, hinge on the precise point where the laser interacts with the sample/matrix crystal. This challenge is somewhat alleviated by averaging a greater number of laser shots across various positions. Nevertheless, the quantitative assessment of MALDI spectra remains a somewhat challenging endeavor [144]. Gangliosides are GSLs possessing one or more attached molecules of sialic acid. Different methods of MALDI MS have been used to characterize these GLSs. On the traditional MALDI-TOF MS, an axial time-of-flight (a-TOF) or orthogonal-TOF (o-TOF) instrument can be employed. Typically, analogous patterns were observed in negative and positive ion modes for both native and methyl-esterified gangliosides using a-TOF and o-TOF analyzers. However, o-TOF exhibited a higher signal intensity and a more favorable S/N ratio for all species, along with enhanced detection accuracy and sensitivity [117]. When employing MALDI MS for the analysis of GSLs, whether in positive or negative ionization mode, fragmentation is a common occurrence [117, 118, 119, 120, 121]. Notably, UV-MALDI led to substantial sialic acid loss, prompting Michiko Tajiri et al. [119] to adopt IR-MALDI at a wavelength of 5.9 \(\upmu\)m. This approach generated deprotonated ions [M - H]\({}^{-}\) and facilitated the more sensitive analysis of negatively charged biomolecules such as intact gangliosides, surpassing the capabilities of UV-MALDI. Another strategy to mitigate fragmentation is the methyl esterification of GSLs, which involves neutralizing all of the carboxylic groups on the sialic acids. Methyl esterification enhances ganglioside stability and simplifies spectra interpretation, leading to more reliable data assignment, particularly within complex biological ganglioside mixtures. The application of o-TOF as an analyzer significantly boosts the stability of sialic acid residues on ganglioside precursors [117]. Penn et al. [121] compared ESI/FTMS with MALDI/FTMS. Negative ion mode gave better results in both procedures. MALDI/FTMS showed slightly more fragmentation. This was caused by differences in internal energy of the ionization process. Fragmentation can also be caused by trapping voltages and quadrupole offset which cause differences in translational energy. Positive ion MALDI/FTMS gave a good signal, but unknown compound identification would be difficult mainly because of multiple metal coordination. Fragmentation in MALDI can be manipulated by using alkali metal doping as well as manipulation of the laser intensity. When using MALDI FTMS, high resolution and accurate mass determination are achievable with this approach, along with the added capability to conduct tandem MS for investigating the structural characteristics of different molecules [131]. Nevertheless, fragmentation can still manifest in this context, although this can be diminished by subjecting the system to pulsed high pressure within the 1-10 mbar range during desorption. In the absence of collisional cooling, the base peak in the spectrum tends to be the asialo fragment, a product of sialic acid loss through fragmentation. However, within the 1-10 mbar range, the base peak transitions to the unfragmented sodiated molecular ion [120]. The task of characterizing ganglioside structures can be particularly challenging, especially when dealing with complex gangliosides harboring numerous sialic acid residues. Methods have been developed to enhance this aspect. TOF/TOF-MS analysis can distinguish between \(\alpha\)2,3- and \(\alpha\)2,8-linked sialic acids, evidenced by distinct fragmentation patterns observed in the TOF/TOF-MS spectra of GD1a and GD1b [149]. In the context of MALDI quadrupole ion trap time-of-flight (QIT-TOF) MS, which enables MSn analysis and sensitive detection through multiple reaction monitoring (MRM), further characterization might necessitate MS, MS2, and/or MS3 analyses.