Glycolipidomics of human cerebellum in development and aging by ion mobility tandem mass spectrometry

In this study ion mobility separation (IMS) mass spectrometry (MS) was for the first time introduced in human cerebellum ganglioside research. The work was focused on a comprehensive mapping and structural characterization of human cerebellar gangliosides and determination of the specific changes induced in their expression by brain development and aging. We have carried out a comparative IMS MS mapping of the native ganglioside mixtures extracted from fetal cerebellum in the second trimester of pregnancy vs. near-term fetus vs. aged cerebellum, followed by IMS CID MS/MS fragmentation analysis

Screening and sequencing of sialylated glycosphingolipids in human glioblastoma by ion mobility mass spectrometry

High performance ion mobility separation mass spectrometry (IMS MS) was thoroughly optimized to allow the discovery of glioblastoma multiforme (GBM)-specific structures and the assessment of their roles as tumor markers or possible associated antigens. Ganglioside (GG) separation by IMS according to the charge state, carbohydrate chain length, degree of sialylation and ceramide composition, led to the identification of no less than 160 distinct components [1], which represents 3 folds the number of structures identified before. The detected GGs and asialo-GGs were found characterized by a high heterogeneity in their ceramide and glycan compositions, encompassing up five Neu5Ac residues. The tumor was found dominated in equal and high proportions by GD3 and GT1 forms, with a particular incidence of C24:1 fatty acids in the ceramide

Advanced mass spectrometry methods for the determination of gangliosides structure and functional interactions

To address the issues of high biological relevance of gangliosides (GGs), mass spectrometry (MS) has lately become a method of choice due to its capability to detect minor species in complex mixtures and the unsurpassed sensitivity. GGs are localized in the cellular membrane, with the ceramide (Cer) rooted in the lipid bilayer and the oligosaccharide chain protruding freely outside the cell, acting as a receptor [1,2]. Here, a complex mixture of GGs extracted from adult human brain was first characterized by MS for a thorough mapping and a detailed structural characterization, and lately, the complex GG mixture was submitted to an interaction assay with the B subunit monomers of cholera toxin (Ctb5). Aliquots of the reaction products were collected after 10 and 30 min and also after 60 min and submitted to MS analysis. Multistage fragmentation by electron transfer dissociation (ETD) and collisioninduced dissociation (CID) completed the assay and provided solid data on the noncovalent biding site at the monomer level

Analysis of Oversulfation in a Chondroitin Sulfate Oligosaccharide Fraction from Bovine Aorta by Nanoelectrospray Ionization Quadrupole Time-of-Flight and Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B- and C-type ions and their respective complementary Y- and Z-type fragment ions

Profiling and structural characterization by mass spectrometry of region-specific gangliosides in brain

Gangliosides (GGs), a particular class of glycosphingolipids ubiquitously found in tissues and body fluids, exhibit the highest expression in the central nervous system, especially in brain. GGs are involved in crucial processes, such as neurogenesis, synaptogenesis, synaptic transmission, cell adhesion, growth and proliferation. For these reasons, efforts are constantly invested into development and refinement of specific methods for GG analysis. We have recently shown that ion mobility separation (IMS) mass spectrometry (MS) has the capability to provide consistent compositional and structural information on GGs at high sensitivity, resolution and mass accuracy. In the present study we have implemented IMS MS for the first time in the study of a highly complex native GG mixture extracted and purified from a normal fetal hippocampus in the 17th gestational week (denoted FH17). The combination of electrospray ionization, ion mobility separation and high resolution mass spectrometry in the negative ionization mode enhanced ganglioside separation based not just on the m/z value, but also on the charge state, the carbohydrate chain length and the degree of sialylation. In the generated driftscope plot (drift time versus m/z), 131 distinct gangliosides characterized by high variability of the oligosaccharide core and diversity of the ceramide moiety were identified with an average mass accuracy of 12.3 ppm. As compared to previous studies where no separation techniques prior to MS were applied, IMS MS technique has not just generated valuable novel information on the GG pattern characteristic for hippocampus in early developmental stage, but also provided data related to the GG molecular involvement in the synaptic functions by the discovery of 25 novel structures modified by CH3COO- . By applying IMS in conjunction with collision induced dissociation (CID) tandem MS (MS/MS), novel GG species modified by CH3COOattachment, discovered here for the first time, were sequenced and structurally investigated in details. The present findings, based on IMS MS, provide a more reliable insight into the expression and role of gangliosides in human hippocampus, with a particular emphasis on their cholinergic activity at this level

Ion mobility mass spectrometry of gangliosides in human brain in health and disease

Gangliosides (GGs), a class of glycosphingolipids, are important biomarkers in early diagnosis of CNS pathologies, being in the focus of our research as potential therapeutic targets [1]. A series of neuropsychiatric disorders are characterized by amnesia and disorientation caused by hippocampal atrophy and diminished cholinergic activity. Based on ion mobility separation mass spectrometry (IMS MS) capability for a reliable glycopattern determination, and the occurrence of neuropsychiatric disorders [2,3], we report here on the improvement of novel and high performance IMS MS method for assessing the GG profile in a highly complex mixture extracted from an adult healthy brain region. The IMS separation of GGs based on charge state, carbohydrate chain length and degree of sialylation led to the detection and identification of over 100 species, the larger number of GGs ever reported before in this particular brain region. Moreover, the obtained data supports the concept of GGs cholinergic activity. Furthermore, by applying IMS MS/MS, novel GG species were structurally investigated in details

Characterization of hybrid chondroitin/dermatan sulfate octasaccharide domains in human brain by ion mobility tandem mass spectrometry

We report here on the introduction of a rapid, highly sensitive and reliable approach in a single run, based on ion mobility separation (IMS), high resolution and tandem MS (MS/MS) by collision-induced dissociation (CID) for compositional and structural elucidation of neural chondroitin sulfate (CS) and dermatan sulfate (DS) domains, which implies the determination of the epimerization and the sulfation code of regular and irregular structures. By IMS MS and CID MS/MS, we were able to characterize in details CS/DS octasaccharides from brain obtained after CS/DS chain depolymerization by chondroitin B lyase and to detect sequences that were never found before in the octasaccharide domains of the investigated CS/DS brain fraction