## 3 MALDI Non-Imaging MS Analysis of GSLs
### The Influence of Matrices Used in MALDI Non-Imaging MS Analysis of GSLs
Utilizing alkali earth metal salts instead of alkali metal salts in the ganglioside sample mixture improved the reliability of isomer differentiation and the intensity of diagnostic ions in positive ion mode of MALDI MS/MS [143]. * **ATT (6-azo-2-thiothymine)**  
Zarei et al. [117] conducted a systematic MS sialylation analysis on a mixture of mouse brain gangliosides, utilizing ATT/DAC (6-azo-2-thiothymine/diammonium citrate) as a matrix. They employed both axial time-of-flight (a-TOF) and orthogonal-TOF (oTOF) instruments in the positive ion mode. The use of an ATT/DAC matrix resulted in improved crystal formation, leading to a superior S/N ratio. Additionally, in another work by Zarei et al. [127], the addition of lithium citrate as a co-matrix for ATT during their LC/MALDI MS analysis of GSLs extracted from human erythrocytes yielded a favorable S/N ratio. This approach enhanced crystal formation in the LC eluent and demonstrated high ionizability of the substrates. When adopting negative ion mode MALDI o-TOF analysis with the ATT/DAC matrix, significant changes in the relative intensities of GM1, GD1, and GT1 species were observed, accompanied by a general increase in S/N for all ionic species. Remarkably higher intensities were seen in GD1 and GT1 ion species, with minimal loss of sialic acid residues [117]. In a separate study, Peter B. O'Connor et al. [120] achieved reduced metastable fragmentation of gangliosides by temporarily increasing the pressure within an MALDI-Fourier transform mass spectrometry (FTMS) source to a range of 1-10 mbar during ionization. This approach decreased fragmentation by shifting the dominant peaks from the asialo fragment at \(m/z\) 1305 to the unfragmented molecular ion at \(m/z\) 1596, particularly when ions were desorbed at high pressure. Similar effects were observed for GD1a, GT1b, GQ1b, GP1b, and GP1c samples derived from human brain tissue. These samples showed decreased metastable fragmentation under high pressure conditions, with increasing the number of sialic acid residues making the molecule more fragile and prone to fragmentation [120]. However, even with the application of methyl esterification, some degree of sialic acid loss persists, albeit to a significantly lesser extent. Notably, the GT1b, GD1a, and GD1b fractions do not undergo dehydration or carbon dioxide loss, resulting in heightened sensitivity for the detection of intact molecular species [117]. * **THAP (trihydroxyacetophenone)** Ruh H. et al. [126] investigated the changes in complex neutral GSLs, including globote-traosylceramide (Gb4 Cer), within a rat model of autosomal dominant polycystic kidney disease (ADPKD). They introduced PrimaDrop as an improved and broadly applicable sample preparation method for automated MALDI MS analysis of lipid extracts. This technique facilitated uniform co-crystallization, allowing relative quantification through indirect TLC\(-\)MALDI-TOF-MS.  
During MALDI-TOF-MS analysis of dried droplet lipid analyte preparations, the formation of rings of large crystal aggregates around the edges of spots was observed across all matrices utilized in the study (ATT, DHB, and THAP). While both DHB and ATT yielded satisfactory spectra quality, the co-crystals formed between the analyte and matrix displayed an uneven distribution, leading to unsuccessful automated data acquisition attempts. This issue persisted despite other attempted crystallization methods, including fast evaporation, two-layer crystallization, and sandwich crystallization. To address this challenge, they devised the PrimaDrop method: a lipid sample is initially deposited onto a grounded steel target and allowed to dry, followed by the application of a concentrated matrix solution (in a rapid-evaporating solvent such as a chloroform/methanol mixture (7/3, _v/v_)) directly onto the previously dried lipid droplets. This approach resulted in a nearly uniform crystal distribution of the matrix, neutral kidney lipid extract, Gb4 Cer standard, and silica-extracted GSLs using ATT, DHB, and THAP matrices.