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.

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.

Chip-nanoelectrospray quadrupole time-of-flight tandem mass spectrometry of meningioma gangliosides: a preliminary study.

A strategy combining high-performance thin layer chromatography (HPTLC), laser densitometry, and fully automated chip-based nanoelectrospray (nanoESIchip) performed on a NanoMate robot coupled to QTOF-MS was developed, optimized, and for the first time applied for mapping and structural identification of gangliosides (GGs) extracted and purified from a human angioblastic meningioma specimen. While HPTLC pattern indicated only seven fractions migrating as GM3, GM2, GM1, GD3, GD1a (nLD1, LD1), GD1b, GT1b, and possibly GD2, due to the high sensitivity, mass accuracy, and ability to ionize minor species in complex mixtures, nanoESIchip-QTOF MS was able to discover significantly more GG species than ever reported in meningioma. Thirty-four distinct glycosphingolipid components of which five asialo, one GM4, nine GM3, two GM2, two GD3, nine GM1, and six GD1 differing in their ceramide compositions were identified. All structures presented long-chain bases with 18 carbon atoms, while the length of the fatty acid was found to vary from C11 to C25. MS screening results indicated also that the diversity of the expressed GM1 structures is higher than expected in view of the low proportions evidenced by densitometric quantification. Simultaneous fragmentation of meningioma-associated GM1 (d18:1/24:1) and GM1 (d18:1/24:0) by MS/MS using CID confirmed the postulated structures of the ceramide moieties and provided data on the glycan core, which document that for each of the GM1 (d18:1/24:1) and GM1 (d18:1/24:0) forms both GM1a and GM1b isomers are expressed in the investigated meningioma tissue.

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry.

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS(2)-MS(3)) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by beta-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Delta-[IdoA-GalNAc]. By optimized CID MS(2)-MS(3), fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Delta-[GlcA-GalNAc]. The site of oversulfation was determined by MS(2)-MS(3), which provided sequence patterns consistent with a rare GlcA-3-sulfate-GalNAc-6-sulfate structural motif. Figure Mouse brain GlcA-3-sulfate-GalNAc-6-sulfate structural motif.

Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry.

We report here on a preliminary investigation of ganglioside composition and structure in human hemangioma, a benign tumor in the frontal cortex (HFC) in comparison to normal frontal cortex (NFC) tissue using for the first time advanced mass spectrometric methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID). The high ionization efficiency, sensitivity and reproducibility provided by the chip-nanoESI approach allowed for a reliable MS-based ganglioside comparative assay. Unlike NFC, ganglioside mixture extracted from HFC was found dominated by species of short glycan chains exhibiting lower overall sialic acid content. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS(2) mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor.

High-throughput analysis of gangliosides in defined regions of fetal brain by fully automated chip-based nanoelectrospray ionization multi-stage mass spectrometry.

Gangliosides (GGs), a large group of sialylated glycosphingolipids, are considered biomarkers of human brain development, aging and certain diseases. Determination of individual GG components in complex mixtures extracted from a human brain represents a fundamental prerequisite for correlating their specificity with the specialized function of each brain area. In the context of modern glycomics, detailed investigation of GG expression and structure in human brain requires a continuous development and application of innovative methods able to improve the quality of data and speed of analysis. In this work, for the first time, a high-throughput mapping and sequencing of gangliosides in human fetal brain was performed by a novel mass spectrometry (MS)-based approach developed recently in our laboratory. Three GG mixtures extracted and purified from different regions of the same fetal brain in the 36th gestational week: frontal neocortex (NEO36), white matter of the frontal lobe (FL36) and white matter of the occipital lobe (OL36) were subjected to comparative high-throughput screening and multi-stage fragmentation by fully automated chip-based nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT) MS. Using this method, in only a few minutes of signal acquisitions, over 100 GG and asialo-GG species were detected and identified in the three mixtures. Obtained data revealed for the first time that differences in GG expression in human fetal brain are dependent on phylogenetic development rather than topographic factors. While a significant variation of GG distribution in NEO36 vs FL36 was observed, no significant differences in GG expression in white matter of frontal vs occipital lobe were detected. Additionally, the largest number of species was identified in NEO36, which correlates with the functional complexity of neocortex as the newest brain region. In the last stage of analysis, using MS(2)-MS(3) molecular ion fragmentation at variable amplitudes, a NEO36-associated GD1b isomer could clearly be discriminated. Present results indicate that the combination of fully automated chipESI with HCT MS(n) is able to provide ultra-fast, sensitive and reliable analyses of complex lipid-linked carbohydrates from which the pattern of their expression and structure in a certain type of bio-matrix can be determined.

Assessment of the molecular expression and structure of gangliosides in brain metastasis of lung adenocarcinoma by an advanced approach based on fully automated chip-nanoelectrospray mass spectrometry.

Gangliosides (GGs), sialic acid-containing glycosphingolipids, are known to be involved in the invasive/metastatic behavior of brain tumor cells. Development of modern methods for determination of the variations in GG expression and structure during neoplastic cell transformation is a priority in the field of biomedical analysis. In this context, we report here on the first optimization and application of chip-based nanoelectrospray (NanoMate robot) mass spectrometry (MS) for the investigation of gangliosides in a secondary brain tumor. In our work a native GG mixture extracted and purified from brain metastasis of lung adenocarcinoma was screened by NanoMate robot coupled to a quadrupole time-of-flight MS. A native GG mixture from an age-matched healthy brain tissue, sampled and analyzed under identical conditions, served as a control. Comparative MS analysis demonstrated an evident dissimilarity in GG expression in the two tissue types. Brain metastasis is characterized by many species having a reduced N-acetylneuraminic acid (Neu5Ac) content, however, modified by fucosylation or O-acetylation such as Fuc-GM4, Fuc-GM3, di-O-Ac-GM1, O-Ac-GM3. In contrast, healthy brain tissue is dominated by longer structures exhibiting from mono- to hexasialylated sugar chains. Also, significant differences in ceramide composition were discovered. By tandem MS using collision-induced dissociation at low energies, brain metastasis-associated GD3 (d18:1/18:0) species as well as an uncommon Fuc-GM1 (d18:1/18:0) detected in the normal brain tissue could be structurally characterized. The novel protocol was able to provide a reliable compositional and structural characterization with high analysis pace and at a sensitivity situated in the fmol range.

Top-down glycolipidomics: fragmentation analysis of ganglioside oligosaccharide core and ceramide moiety by chip-nanoelectrospray collision-induced dissociation MS2-MS6.

We developed a straightforward approach for high-throughput top-down glycolipidomics based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) multistage mass spectrometry (MSn) by collision-induced dissociation (CID) in the negative ion mode. The method was optimized and tested on a polysialylated ganglioside fraction (GT1b), which was profiled by MS1 and sequenced in tandem MS up to MS6 in the same experiment. Screening of the fraction in the MS1 mode indicated the occurrence of six [M-2H]2- ions which, according to calculation, support 13 GT1 variants differing in their relative molecular mass due to dissimilar ceramide (Cer) constitutions. By stepwise CID MS2-MS5 on the doubly charged ion at m/z 1077.20 corresponding to a ubiquitous GT1b structure, the complete characterization of its oligosaccharide core including the identification of sialylation sites was achieved. Structure of the lipid moiety was further elucidated by CID MS6 analysis carried out using the Y0 fragment ion, detected in MS5, as a precursor. MS6 fragmentation resulted in a pattern supporting a single ceramide form having the less common (d20 : 1/18 : 0) configuration. The entire top-down experiment was performed in a high-throughput regime in less than 3 min of measurement, with an analysis sensitivity situated in the subpicomolar range.