Chip-based high resolution tandem mass spectrometric determination of fibroblast growth factor-chondroitin sulfate disaccharides noncovalent interaction.

Fibroblast growth factor-2 (FGF-2) is involved in wound healing and embryonic development. Glycosaminoglycans (GAGs), the major components of the extracellular matrix (ECM), play fundamental roles at this level. FGF-GAG noncovalent interactions are in the focus of research, due to their influence upon cell proliferation and tissue regeneration. Lately, high resolution mass spectrometry (MS) coupled with chip-nanoelectrospray (nanoESI) contributed a significant progress in glycosaminoglycomics by discoveries related to novel species and their characterization. We have employed a fully automated chip-nanoESI coupled to a quadrupole time-of-flight (QTOF) MS for assessing FGF-GAG noncovalent complexes. For the first time, a CS disaccharide was involved in a binding assay with FGF-2. The experiments were conducted in 10 mM ammonium acetate/formic acid, pH 6.8, by incubating FGF-2 and CS in buffer. The detected complexes were characterized by top-down in tandem MS (MS/MS) using collision induced-dissociation (CID). CID MS/MS provided data showing for the first time that the binding process occurs via the sulfate group located at C4 in GalNAc. This study has demonstrated that chip-MS may generate reliable data upon the formation of GAG-protein complexes and their structure. Biologically, the findings are relevant for studies focused on the identification of the active domains in longer GAG chains.

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.

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.

ß-Lactoglobulin detected in human milk forms noncovalent complexes with maltooligosaccharides as revealed by chip-nanoelectrospray high-resolution tandem mass spectrometry.

Cow's milk protein allergy in exclusively breastfed infants, the main cause of food intolerance during the first 6 months of life, is triggered by the mother's diet. ß-Lactoglobulin (BLG) present in cow's milk is one of the most potent allergens for newborns. Since no prophylactic treatment is available, finding ligands capable of binding BLG and reducing its allergenicity is currently the focus of research. In this work, an innovative methodology encompassing microfluidics based on fully automated chip-nanoelectrospray ionization (nanoESI), coupled with high-resolution mass spectrometry (MS) on a quadrupole time-of-flight (QTOF MS) instrument was developed. This platform was employed for the assessment of the noncovalent interactions between maltohexaose (Glc6) and ß-lactoglobulin extracted from human milk upon deliberate intake of cow's milk. The experiments were carried out in (+) ESI mode, using ammonium acetate (pH 6.0) as the buffer and also in pure water. In both cases, the MS analysis revealed the formation of BLG-Glc6 complex, which was characterized by top-down fragmentation in tandem MS (MS/MS) using collision-induced dissociation (CID). Our findings have a significant biomedical impact, indicating that Glc6 binds BLG under conditions mimicking the in vivo environment and therefore might represent a ligand, able to reduce its allergenicity.

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.

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.