Adrian C. Robu

Electrospray Ionization Ion Mobility Mass Spectrometry of Human Brain Gangliosides

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.

Mass spectrometry of gangliosides in extracranial tumors: Application to adrenal neuroblastoma.

We report here on the introduction of mass spectrometry (MS) for profiling of native gangliosides from an extracranial tumor. The analytical approach was based on a modern platform combining the superior sensitivity and reproducibility of fully automated chip-based nanoelectrospray ionization (nanoESI) with the high resolution and mass accuracy provided by a hybrid quadrupole time-of-flight (QTOF) instrument. The feasibility of the method for the analysis of gangliosides, which are much less expressed in extracranial tissues, was here tested using as the model substrate an adrenal neuroblastoma (NB) specimen located in the abdominal region of a 2-year-old infant. Under properly optimized conditions, MS profiling revealed information on at least 61 different gangliosides exhibiting heterogeneity of the glycan and lipid compositions. NB was found dominated by species bearing short-chain oligosaccharide cores with a reduced overall Neu5Ac content. By chip-nanoESI MS, preceding findings related to the GD2 role in NB were confirmed. Moreover, the screening experiments offered novel information supporting the possible biomarker role of GM4, GM3, and GM1 ganglioside classes. Structural analysis of GM1(d18:1/18:2) and GD1(d18:0/19:0) possibly tumor-associated markers, carried out by tandem MS (MS/MS) using collision-induced dissociation (CID) at low energies, indicated that both GM1a and GD1b isomers are present in NB.

Automated chip-nanoelectrospray mass spectrometry for glycourinomics in Schindler disease type I.

In this study an integrative mass spectrometry (MS) approach based on fully automated chip-nanoelectrospray quadrupole time-of-flight was optimized and applied for the discovery and structural characterization of O-glycopeptides in a fraction from the urine of a patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides extracted and purified from an age matched healthy subject served as the control. 49 glycoforms were discovered in the investigated urine fraction from Schindler disease versus only 14 in control urine. Structures with relevant biological significance, previously not described, such as O-fucosylated tetrasaccharides and chains up to pentadecamers O-linked to serine, threonine, or threonine-proline were identified in the pathological urine and characterized by tandem MS (MS/MS). A number of 29 species discovered here, most of which with long chain glycans, were not previously reported as associated to this condition. All glycopeptides were detected in only 1 min analysis time, with a sample consumption situated in the femtomole range.

Orbitrap mass spectrometry characterization of hybrid chondroitin/dermatan sulfate hexasaccharide domains expressed in brain

In the central nervous system, chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) modulate neurotrophic effects and glial cell maturation during brain development. Previous reports revealed that GAG composition could be responsible for CS/DS activities in brain. In this work, for the structural characterization of DS- and CS-rich domains in hybrid GAG chains extracted from neural tissue, we have developed an advanced approach based on high-resolution mass spectrometry (MS) using nanoelectrospray ionization Orbitrap in the negative ion mode. Our high-resolution MS and multistage MS approach was developed and applied to hexasaccharides obtained from 4- and 14-week-old mouse brains by GAG digestion with chondroitin B and in parallel with AC I lyase. The expression of DS- and CS-rich domains in the two tissues was assessed comparatively. The analyses indicated an age-related structural variability of the CS/DS motifs. The older brain was found to contain more structures and a higher sulfation of DS-rich regions, whereas the younger brain was found to be characterized by a higher sulfation of CS-rich regions. By multistage MS using collision-induced dissociation, we also demonstrated the incidence in mouse brain of an atypical [4,5-Δ-GlcAGalNAc(IdoAGalNAc)2], presenting a bisulfated CS disaccharide formed by 3-O-sulfate-4,5-Δ-GlcA and 6-O-sulfate-GalNAc moieties.

B subunit monomers of cholera toxin bind G1 ganglioside class as revealed by chip- nanoelectrospray multistage mass spectrometry

GRAPHICAL ABSTRACT The noncovalent interaction between the monomers (Ctb) of the B subunit (Ctb5) of cholera toxin from Vibrio cholerae and a native complex mixture of gangliosides extracted and purified from adult human cerebellum was studied using an analytical platform encompassing fully automated chip-nanoelectrospray ionization (nanoESI) on a NanoMate robot coupled to a high-capacity ion trap (HCT) mass spectrometer (MS). The interaction assay involved the incubation of Ctb5 and gangliosides dissolved in 10 mM ammonium acetate buffer at pH 5.8. This pH value enhanced the dissociation of the monomers constituting Ctb5 and allowed the direct interaction between the monomers and gangliosides. Aliquots of the reaction products were collected directly after 10, 30, and 60 min of incubation in the 96-well plate of the NanoMate robot and immediately submitted to MS analysis. Except for the Ctb envelope, positive ion mode chip-nanoESI mass spectra presented 25 signals corresponding to multiply protonated ions of the Ctb complexes with different gangliosides, all belonging to the G1 class. Certain complexes revealed Ctb interaction with G1 species of high sialylation degree or modified by fucosylation, which were not discovered in human cerebellum before. Multistage fragmentation carried out by electron transfer dissociation (ETD) followed by collision-induced dissociation (CID) on the Ctb complex with Fuc-GT1 (d18:1/18:0) provided data supporting the binding of the c isomer of Fuc-GT1 (d18:1/18:0) to histidine 14 (H14), most probably via Neu5Ac.

Advances in Mass Spectrometry for Glycoscreening and Sequencing in Biomedical Research

During the past several years, applications of Mass Spectrometry (MS) in the biomedical research increased considerably. While MS is for many years heavily used in proteomics, for protein identification and quantification as well as for biomarker discovery, for a long time its applications in glycomics were limited, mainly because of the challenging conditions required for the ionization and detection of most carbohydrate classes. However, due to the development of high performance analytical instrumentation, MS in particular with Electrospray (ESI) and Matrix Assisted Laser Desorption/Ionization (MALDI) started to be intensively applied also to the analysis of post-translational modifications such as glycosylation, acetylation or phosphorylation. Focus on MS-based glycosylation is, however, scarce. Therefore, analysis of glycoproteins in particular diseases through glycoscreening and sequencing is another new MS-based avenue, yet to be pursued. In this context, we discuss briefly here the recent advances of MS in glycomics and glycoscreening and their applications in biomedical research, with a particular emphasis on cancer, lysosomal storage and bacterial diseases.

Actual trends in mass spectrometry of glycosaminoglycans

Abbreviations: CE=capillary electrophoresis; CS=chondroitin sulfate; DS=dermatan sulfate; ECM=extrace llular matrix; EDD=electron detachment dissociation; ESI=electrospray ionization; FAIMS=high-fi eld asymmetric waveform ion mobility spectrometry; FTICR=Fourier transform ion cyclotron resonance; FTMS=Fourier transform mass spectrometry; GAG=glycosaminoglycan; GalNAc=N-acetylgalactosamine; GlcA=glucuronic acid; HexA=hexuronic acid; HILIC=hydrophilic interaction chromatography; HS=heparan sulfate; IdoA=iduronic acid; IMS=ion mobility spectrometry; KS=keratan sulfate; LC=liquid chromatography; MALDI=matrix assisted laser desorption/ionization; MS/MS=tandem mass spectrometry; MW=molecular weight; NETD=negative electron transfer dissociation; PG=proteoglycan; RF=radio fi re; SEC=size-exclusion chromatography; SPE=solid phase extraction; UA=uronic acid