Electrospray Ionization Ion Mobility Mass Spectrometry of Human Brain Gangliosides

Abstract

The progress of ion mobility spectrometry (IMS), together with its association to mass spectrometry (MS), opened new directions for the identification of various metabolites in complex biological matrices. However, glycolipidomics of the human brain by IMS MS represents an area untouched up to now, because of the difficulties encountered in brain sampling, analyte extraction, and IMS MS method optimization. In this study, IMS MS was introduced in human brain ganglioside (GG) research. The efficiency of the method in clinical glycolipidomics was demonstrated on a highly complex mixture extracted from a normal fetal frontal lobe (FL37). Using this approach, a remarkably rich molecular ion pattern was discovered, which proved the presence of a large number of glycoforms and an unpredicted diversity of the ceramide chains. Moreover, the results showed for the first time the occurrence of GGs in the human brain with a much higher degree of sialylation than previously reported. Using IMS MS, the entire series starting from mono- up to octasialylated GGs was detected in FL37. These findings substantiate early clinical reports on the direct correlation between GG sialylation degree and brain developmental stage. Using IMS CID MS/MS, applied here for the first time to gangliosides, a novel, tetrasialylated O-GalNAc modified species with a potential biomarker role in brain development was structurally characterized. Under variable collision energy, a high number of sequence ions was generated for the investigated GalNAc-GQ1(d18:1/18:0) species. Several fragment ions documented the presence of the tetrasialo element attached to the inner Gal, indicating that GalNAc-GQ1(d18:1/18:0) belongs to the d series.

Orbitrap mass spectrometry for monitoring the ganglioside pattern in human cerebellum development and aging

“…The C15gw, C40gw, and C65y ganglioside mixtures were extracted and purified following the protocol described in Almeida et al The protocol was developed by the group of Vukelić and applied by us previously . Briefly, the tissue was homogenized in ice‐cold water.…”

“…All these features emphasize once more the importance of this type of glycoconjugates at the brain level. For these reasons, in the last years, several innovative approaches focused on the analysis of fetal and adult human brain ganglioside composition, structure, and functional interactions, most of them involving highly sensitive MS techniques, were developed . In the particular case of human cerebellum, some distinctive variations in the composition of major ganglioside classes in comparison to the cerebrum as well as the correlation between ganglioside sialylation degree and the developmental stage of fetal cerebellar tissue were postulated by our groups on the basis of the data collected by fully automated chip‐nanoESI MS on a NanoMate robot coupled to either a quadrupole‐time‐of‐flight (QTOF) MS or a high capacity ion trap (HCT) MS instrument …”

Structural Analysis of Unsaturated Glycosphingolipids Using Shotgun Ozone-Induced Dissociation Mass Spectrometry

“…A wealth of evidences further suggests the importance of unsaturation in the overall attributes of these molecules. Besides its conspicuous effect on membrane fluidity [11], unsaturated glycosphingolipids are also present in specialized tissues/cells such as brain [12], retina[13], and sperm [14] thus indicating that olefin in the lipid backbone is an essential motif for highly specialized biological function. The involvement of lipids and glycolipids as antigens has been demonstrated in prior studies further augmenting the relationship between unsaturation and immunogenic behavior.…”

“…Specifically, different double bond positions could result in distinct structural conformations thus influencing antigenicity and ultimately, recognition by immune cells [15–19]. Studies also show that some of the potential glycosphingolipid biomarkers of brain development and the underlying diseases are in fact, unsaturated, yet rarely are they identified down to the finest structural detail [12, 20–23]. As such, comprehensive structural determination of intact glycosphingolipids is warranted.…”

Quantitative Screening of Cell‐Surface Gangliosides by Nondestructive Extraction and Hydrophobic Collection

“…This technology can determine their molecular structures. [9,10] Some chromatographic and immunochemical techniques have also been developed for the detection of primary species of major gangliosides. [11,12] However,t hese methods need to destroy tissues or cells to separate gangliosides through complicated procedures,which leads to al oss of target information.…”