## 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
Gangliosides were isolated from various types of human tissues (leukocytes, erythrocytes, small intestine, stomach, meconium, colon, pancreas), but leukocytes showed the best binding capacity of gangliosides in the assay conditions and were further analyzed. Human white blood cell mixtures were obtained from healthy donors' venous blood. The buffy coats, comprising of polymorphonuclear leukocytes, were isolated by lysing erythrocytes using a 0.8% NH4 Cl solution. After 400\(\times\)\(g\) centrifugation, fractions containing 70% to 85% polymorphonuclear leukocytes were selected for further experiments. To facilitate the analysis, the gangliosides underwent mild periodate oxidation. They were incubated with NaIO4 (1-2 mM) in acetate buffer (pH 5.5) on ice for a duration of 40 min, followed by the cessation of the reaction through the addition of an excess amount of Na2 SO3. The resultant mixture underwent freeze-drying, leading to a concentration increase of approximately five-fold. Next, the sample was subjected to reduction using an excess of NaBH4 at room temperature over the course of an overnight period. Subsequent to this, the sample was subjected to dialysis using distilled water, and the final step involved freeze-drying. For specific binding assays, TLC overlay techniques were employed. Different overlays were conducted (influenza virus overlay, antibody overlay, and lectin overlay)  
The human influenza virus exhibited a highly selective binding profile, primarily targeting extended glycolipids found in human leukocytes. Notably, it displayed negligible binding affinity to shorter ganglioside species, including the commonly abundant GSL species with 6-linked NeuAc. Weak binding interactions were also observed with species which are slow-moving derived from various human tissues such as the small intestine and pancreas. In contrast, the avian influenza virus displayed broader binding capabilities, interacting with various gangliosides across all tested tissues. It showed an affinity for species which are NeuAc\(\alpha\)3 Gal-terminated, as evidenced by its binding to NeuAc\(\alpha\)3-paragloboside (S-3-PG) and GD1a and GT1b gangliosides [183]. The isolated gangliosides, particularly those derived from leukocytes, were subjected to MALDI-TOF MS analysis. The MALDI-TOF mass spectra revealed the presence of complex gangliosides with 8-11 monosaccharides per mol of Cer, as follows: \(m/z\) 2394.0 (NeuAc\({}_{1}\)Fuc\({}_{1}\)Hex\({}_{3}\)HexNA\({}_{\rm C3}\)Cer--d18:1/C16:0), \(m/z\) 2248.7 (NeuAc\({}_{1}\)Hex\({}_{5}\)HexNA\({}_{\rm C3}\)Cer--d18:1/C16:0), \(m/z\) 2504.4 (NeuAc\({}_{1}\)Fuc\({}_{1}\)Hex\({}_{5}\)HexNA\({}_{\rm C3}\)Cer d18:1/C24:1), \(m/z\) 2173.7 (NeuAc\({}_{2}\)Hex\({}_{4}\)HexNAc\({}_{2}\)Cer d18:1/C16:0 and/or NeuAc\({}_{1}\)Fuc\({}_{2}\)Hex\({}_{4}\)HexNA\({}_{\rm C2}\)Cer d18:1/C16:1), \(m/z\) 2649.3 (NeuAc\({}_{2}\) Hex\({}_{5}\)HexNA\({}_{\rm C3}\)Cer d18:1/C24:1 and/or NeuAc\({}_{1}\)Fuc\({}_{2}\)Hex\({}_{5}\)HexNAAc\({}_{\rm C3}\)Cer d18:1/C24:1 and/or NeuAc\({}_{1}\)Fuc\({}_{2}\)Hex\({}_{5}\)HexNA\({}_{\rm C3}\)Cer d18:1/C24:2), and at \(m/z\) 2030.4 (NeuAc\({}_{1}\)Fuc\({}_{1}\)Hex\({}_{4}\)HexNA\({}_{\rm C2}\)Cer d18:1/C16:0). The most abundant molecular ions corresponded to gangliosides such as NeuAc\({}_{1}\)Fuc\({}_{1}\)Hex\({}_{5}\)HexNAc\({}_{\rm C3}\)Cer and NeuAc\({}_{1}\)Hex\({}_{5}\)HexNA\({}_{\rm C3}\)Cer. The authors discovered that the human influenza virus selectively binds to complex fractions found in human leukocytes. Sialyl Lewis X and VIM-2-active saccharides were ruled out as binding sequences. The binding was dependent on the intact glycerol sialic acid tail and involved minor NeuAc\(\alpha\)6-containing species. Overlapping binding with anti-silyl Lewis x antibodies but not with less complex gangliosides was also noted.