## 3 MALDI Non-Imaging MS Analysis of GSLs
### MALDI Non-Imaging MS Analysis of GSLs in the Healthy State
#### 3.4.2. GSLs from Human Brain Tissues  
In the human brain, gangliosides have a multidimensional and complex function, and research is ongoing for further understanding of their specific roles in neural function and disease. They are abundant in the myelin sheath that surrounds the neurons, taking part in functions such as neuronal signaling (modulating the activity of neurotransmitters), synapse formation, cell to cell interaction, and of course, formation of the myelin sheath [156]. In regards to nervous system disorders, gangliosides have been found to play a part in the development of Parkinson's disease, Alzheimer's disease, multiple sclerosis, as well as some types of cancer [135; 136; 137; 138; 139; 140; 141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152; 153; 154; 155; 156; 157; 158; 159; 160; 161; 162]. Due to the limited accessibility to samples of normal human brain tissue, there has been a notable dearth of research endeavors addressing this particular concern. Human brain tissue samples (cerebellar, cerebrum and cortex, and gray matter from the occipital lobe) [163; 164] are used alongside ganglioside standards to develop a technique for detecting molecular ion species without losing the labile sialic acid residues, using MALDI FT MS with ATT as a matrix by O'Connor et al. [120]. Following the crude lipid extraction from the human brain sample, two successful silica gel column migrations were performed for obtaining a ganglioside-enriched fraction. The second ganglioside-enriched fraction was subjected to alkaline hydrolysis, dialysis, and underwent HPTLC (bromophenol blue was used as color indicator), thus obtaining the purified ganglioside fraction that was subsequently subjected to acetylation (formamide/pyridine/acetic anhydride), dilution, and dialysis, applied to a Sephadex G25 chromatography column and a final silica gel column, from which the second eluate contained the highly purified ganglioside [164]. The research results indicate that with an increasing count of residues of sialic acid, the ion species exhibit heightened vulnerability, rendering them susceptible to fragmentation through the loss of carboxyl groups and sialic acid, resulting in the formation of formic acid. However, under collisional cooling conditions, the unfragmented sodiated molecule remains dominant as the highest intensity peak [120]. During the study, non-covalent matrix adduction was observed, forming ATT matrix clusters. The S/N ratio was higher in the human brain tissue sample compared to the ganglioside standards. Compared to the ganglioside standard, the human brain GSL mixture (GP1b,1c and GQ1b) expressed, under high pressure, diminished metastable fragmentation in a significant manner. Both GQ1b (at \(m/z\) 2512.12 \(\left[\mathrm{M}(3\mathrm{Na})-\mathrm{H}\right]^{-}\) and GP1b,1c (at \(m/z\) 2798.13 \(\left[\mathrm{M}(4\mathrm{Na})-\mathrm{H}\right]^{-}\) were obtained in negative ion mode, proving that coupling TLC separations with MALDI-FTMS permits the analysis of gangliosides without fragmentation, providing high accuracy and resolution [120]. DE (delayed ion extraction) MALDI-TOF MS (Voyager Elite XL Biospectrometry Workstation (PerSeptive Biosystems, Framingham, MA, USA) with nitrogen laser pulsed at 337 nm wavelength) was used by Sugiyama et al. [162] for identifying several gangliosides from normal (GD1a, GD1b, GD2, GalNAc-GD1a) and pathological brain tissues (detailed in the next subsection). Both ion modes, negative and positive, were employed. Using both A-CHCA and 2.5 DHB as matrices, and both ion modes (negative and positive) for establishing a suitable protocol, the authors used a method of microwave-mediated saponification for the gangliosides for preparing their lysogangliosides in order to determine the ceramide composition [123; 128]. The method is used for easier determination of sphingoid base GSL composition. Comparing the obtained spectra with the calculated values of monoisotopic molecular ions, the obtained results were promising.