## 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  
GSLs play vital roles in signal transduction, cell-to-cell communication, and the membrane structure in blood cell components, having several important functions essential for the proper functioning of the immune system, blood homeostasis, the process of blood clotting, and hemostasis [32]. Three major GSL species were identified in erythrocytes: lactosylceramide (LacCer), globotriaosylceramide (Gb3 Cer), and globotetraosyliloceramide (Gb4 Cer), each with C16 and C24 fatty acid chains. [127]. In red blood cells, GSLs act as blood group antigens (globo-family GSLs proceed from the addition of an \(\alpha 1\to 4\)Gal to LacCer to form globotriaosylceramide (Gb3, Pk), which will then serve as a substrate for the formation of the globoside (Gb4, P antigen) by attaching a terminal \(\beta 1\to 3\)GalNAc to Gb3), but also have been shown to play a role in the migration and adhesion of blood cells [171]. Zarei et al. [127] carried out an analysis of GSLs extracted from human erythrocytes. Anion-exchange chromatography was used to isolate neutral GSLs, which were then purified by silica gel gradient chromatography. Afterward, the GSLs were peracetylated and separated on Florisil, eluted with dichloroethane/acetone, saponified, and desalted, the obtained mixture of neutral GSLs was adjusted to a concentration of 1 \(\mu\)g/\(\mu\)L. A nano-HPLC/MALDI MS coupling was achieved (Ultimate nanoLC system with Famos autosampler and nanoscale TSK-GEL Amide-80 column; MALDI-TOF Reflex III, Bruker Daltonics, with nitrogen laser pulsed at 337 nm and DHB as matrix), while for MALDI spotting, a Proteineeer FC robot from Bruker Daltonics (Bruker Daltonics, Bremen, Germany) was used and the best reproducible matrix deposition with efficient crystallization was a DHB matrix dissolved in acetonitrile/water (7:3, \(v/v\)). Depending on the ionization requirements of the analytes, both negative and positive ion modes were operated on the instrument. Three major GSL species were identified: lactosylceramide (LacCer), globotriaosylcle-ramide (Gb3 Cer), and globotetraosyliloceramide (Gb4 Cer), each with fatty acid chains C16 and C24. According to the elution times, LacCer was identified at \(m/z\) 884.79 (d18:1 C16:0) and 996.78 (d18:1/C24:0) at 38 min and at \(m/z\) 884.67 (d18:1/C16:0) at 43 min; Gb3 Cer was only identified at \(m/z\) 1158.71 (d18:1/C24:0) and Gb4 Cer, the dominant GSL according to the intensities, was identified at \(m/z\) 1249.83 (d18:1/C16:0), 1333.86 (d18:1/C22:0), 1359.89 (d18:1/C24:1), and 1361.88 (d18:1/C24:0). In order to suppress the formation of cation adducts of different kinds (H\({}^{+}\), K\({}^{+}\), Na\({}^{+}\)), the authors proposed adding lithium salts for convenient analysis. After a trial-and-error process, the ATT matrix with lithium citrate as a co-matrix was chosen as the most suitable combination. In the mass spectra, the GSL species were exclusively identified in their lithiated forms, at elution times of 41 min and 43 min, respectively, for Gb3 Cerat \(m/z\) 1140.68 \(\left[\text{Gb}3(18:1/24:1)+\text{Li}\right]^{+}\), \(m/z\) 1142.71 \(\left[\text{Gb}3(18:1/24:0)+\text{Li}\right]^{+}\) and for Gb4 at \(m/z\) 1233.65 \(\left[\text{Gb}4(18:1/16:0)+\text{Li}\right]^{+}\), \(m/z\) 1317.76 \(\left[\text{Gb}4(18:1/22:0)+\text{Li}\right]^{+}\), \(m/z\) 1343.83 \(\left[\text{Gb}4(18:1/24:1)+\text{Li}\right]^{+}\), and at \(m/z\) 1345.82 \(\left[\text{Gb}4(18:1/24:0)+\text{Li}\right]^{+}\). The protocol is suggested for further analysis, as it is simple and minimizes sample loss by crystallization procedures. In red blood cells, GSLs act as blood group antigens (globo-family GSLs are obtained from the addition of an \(\alpha 1\to 4\)Gal to LacCer to form globotriasolceramide (Gb3, Pk), which will then serve as a substrate for the formation of the globoside (Gb4, P antigen) by attaching a terminal \(\beta 1\to 3\)GalNAc to Gb3), but also have been shown to play a role in the migration and adhesion of blood cells [171].