## 3 MALDI Non-Imaging MS Analysis of GSLs
### MALDI Non-Imaging MS Analysis of GSLs in the Healthy State
#### 3.4.3. GSLs Extracted from Human Blood Cells
The oligoglycoslyceramides of erythrocytes--known as blood-type determinants--have attracted special interest, as they are important for cellular interactions and are essential factors in deciding the suitability of blood and other tissues for transplantation. Moreover, Gb3 was shown to bind specifically to the verotoxins of _Escherichia coli_ and to Shiga-like verotoxins, and it is an important factor in the progress of disease by enabling access into cells. Gb3 has been involved with other GSLs in host-cell interactions with the HIV virus, where it appears to have a protective role, while Gb4 helps the entry of the parvovirus B19 into cells. B19 parvovirus causes erythema infectiousum and leads to congenital anemia and hydrops fetalis subsequent to in utero infection. It is also linked to transient aplastic crisis in patients with hemolytic anemia and with cases of pure erythrocyte aplasia and chronic anemia in immunocompromised individuals. Gb3 levels and levels of particular gangliosides, are increased in some cancers, and are connected with an increase in multidrug resistance of the tumor [172; 173]. Regarding human platelets, Taketomi et al. [162] proposed an analysis performed with DE MALDI-TOF MS, using a Voyager Elite XL (PerSeptive Biosystem, Framingham, MA, USA), equipped with a nitrogen laser (337 nm) and using 2, 5 DHB as a matrix. Using the preparation of GSLs through the method of saponification [162; 163] described earlier, the mass spectrum revealed molecular species of sphingomyelin, GlcCer (glucoseylateamide), LacCer, Gb3 Cer, Gb4 Cer, and GM2, with sphingomyelin being the major sphingolipid detected [162]. Another study [174] revealed that LacCer and GM3 are the major GSLs of human platelets, while GD3 was synthesized upon platelet activation and selectively stimulated human platelet adhesion, spreading, and aggregation [175]. In addition, GalCer [176], sulfatides [177], GlcCer [178], GM1 [179], Gb3 [180], and sially-galactosylgloboside [181] have also been found in human platelets. In platelets, GSLs are involved in the aggregation of platelets, adhesion to the injured blood vessels, signal transduction, and thrombus formation [174; 182]. This method permits the detection of multiple GSLs from a unique sample, with quick steps for sample preparation, offering perspectives on the future use of MS in GSL profiling from human blood samples. A small quantity of platelets (approximately 50 mg) is employed, which is particularly advantageous when dealing with limited sample volumes. While using chloroform:methanol (2:1, \(v/v\)) as the extraction solvent mixture ensures efficient extraction of lipid components, the stepwise extraction process enables the selective removal of specific lipid classes, such as cholesterol, simple lipids, and glycerophospholipids, thereby enhancing the specificity of the analysis. Moreover, the saponification step involving 0.5 N NaOH in methanol at 50 \({}^{\circ}\)C for 1 h facilitates the breakdown of complex lipids, allowing for the individual identification of molecular species. Subsequently, the separation of the upper and lower phases, followed by centrifugation, aids in the removal of salts, ensuring a cleaner sample for analysis. MALDI-TOF MS is employed for the precise identification/characterization of various sphingolipids and GSLs. It enables the comprehensive confirmation of major sphingolipids, including sphingomyelin, and the definitive identification of multiple minor GSLs. Miller-Podraza et al. [159; 183] investigated how avian and human influenza viruses bind to gangliosides obtained from diverse human tissues and cells, with a particular focus on leukocytes. MALDI-TOF MS was performed on a TofSpec-E (Micromass, Manchester,UK) using ATT as a matrix, to elucidate the structural characteristics of gangliosides and identify the specific ganglioside species responsible for virus binding.