## 3 MALDI Non-Imaging MS Analysis of GSLs
### MALDI Non-Imaging MS Analysis of GSLs in the Healthy State
#### 3.4.1 GSLs in Human Milk  
Milk is a fluid produced by the human body in order to supply the newborn with all the necessities within the first years of life, however, the importance of the gangliosidesin its structure is not fully understood at the moment [153]. Gangliosides in milk are found entirely in the membrane of the milk fat globule, playing different roles, including modulation of membrane protein function, host-pathogen interactions, and cell-to-cell recognition [122]. Due to their structure (the oligosaccharide headgroup interacts with cells' external environment, while the ceramide moiety anchors within the membrane), gangliosides are considered bioactive components. Aside from its nutritional role, it is known that milk plays a vital role in forming the immunity, metabolism, and modulating the newborn's microflora [122]. The role played by gangliosides found in human milk is still under research, and by determining their structures, as well as comparing this information with samples from other mammalians, new steps are being made in this research. Lee et al. [122] described a rapid method to characterize milk gangliosides using MALDI FITCR MS with a laser pulse at 355 nm, using a standard DHB matrix, in both the negative mode and the positive mode. The ions of interest were further analyzed by tandem MS, using collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). By analyzing bovine milk GM3 and GD3 standards first, the general pattern of fragmentation was identified, with the observation that IRMPD produced less ion fragmentation than CID, and less fragmentation was obtained using negative mode. Using the same experimental conditions, human and bovine milk gangliosides were prepared and analyzed, identifying GM3 (NeuAc-2 Hex-Cer) from \(m/z\) 1152 to 1278 and GD3 (2 NeuAc-2 Hex-Cer) from \(m/z\) 1471 to 1577. Ten GM3 and six GD3 variants were abundantly present ions in human milk [2], suggesting that a high complexity of ganglioside structures can be found in human milk. Seven variants of GM3, and six of GD3, as well as other gangliosides, with the assigned structure 1 NeuAc + 2 HexNac + 4 Hex and variations in the ceramide subspecies were found in bovine milk. Only human milk gangliosides have been found to have the ceramide carbons even-numbered (d34:1, d36:1, d38:1, d40:1, and d42:1). Similar results were obtained by Bode et al. [109] using MALDI TOF-MS performed on a Voyager-DE-STR system, using a 337 nm laser wavelength, also on a DHB matrix, in positive ion mode, when comparing human and bovine milk. A high sodium content was found in the human milk gangliosides, with the detection of double sodium adduct ions on three separate GM3 variations. Bode et al. [109] found that the main fatty acid forms of GM3 in human milk were 22:0, 18:0, 16:0, and 24:0, and in GD3 they were 18:0, 16:0, 19:0, and 22:0. Another study [154] emphasized that long-chain FAs are found in higher abundance in human milk when compared to bovine milk, and higher concentrations of cerebrosides than LacCer also. Regarding the bovine milk, Bode et al. [118] mention that the attribution of mass distributions was from 1176 to 1326 Da for GM3 and from 1523 to 1624 Da for GD3 in bovine milk, and both GM3 and GD3 have mainly 23:0, 22:0, and 24:0 as fatty acids, with an emphasis on the high content of 23:0 fatty acid content from bovine milk, compared with its minor representation in human milk [118]. Both studies compared bovine and human milk obtaining somewhat similar results, with abundant GM3 and GD3 species and higher complexity of their ceramide composition being found in human milk, and GD3 being present in a smaller proportion than in bovine milk, but the biological significance of this pattern is still to be elucidated.