Ganglioside Mixture Research Articles

MO635: Early Diabetic Kidney Disease in Type 2 Diabetes Mellitus Patients is Associated with A Particular Ganglioside Profile, Identified by High-Resolution Tandem Mass Spectrometry:A Pilot Study

Abstract BACKGROUND AND AIMS Metabolic biomarkers are of high importance for the identification of early renal involvement in the course of type 2 diabetes mellitus (DM). Gangliosides, which are complex cell type-specific glycosphingolipids, consist of a sialylated oligosaccharide chain attached to a ceramide of different composition with respect to the sphingoid base and fatty acid residues [1]. Gangliosides play a major role in the pathogenesis of diabetic kidney disease (DKD). The aim of the study was to assess the pattern of urinary gangliosides in patients with type 2 DM. METHOD The urine gangliosidome of 30 type 2 DM patients (10 normo-, 10 micro-, and 10 macroalbuminuric) has been investigated in a cross-sectional pilot study, by a comparative assay with 10 healthy controls. Following the optimization of urine ganglioside extraction procedure [2, 3], we have determined the composition of native ganglioside mixtures from the 24-h collected urine samples using a modern bioanalytical platform based on nanoelectrospray ionization (nanoESI) high-resolution mass spectrometry (HR MS) on an Orbitrap instrument. RESULTS HR MS screening and fragmentation analysis by tandem MS revealed that: (i) the urinary gangliosidome of type 2 DM patients contains a significantly higher number of distinct species differing in either their glycan or ceramide structure than the controls; (ii) the ganglioside urinary level was significantly increased in normoalbuminuric patients compared with healthy control subjects; (iii) the gangliosidome of macroalbuminuric patients is characterized by an elevated overall sialic acid content than normo- and microalbuminuric diabetics and more complex structures, including fucosylated, O-GalNAc- and CH3COO--modified compounds; (iv) the degree of sialylation of species presented correlations with the level of albuminuria and renal function; (v) further detailed structural analysis of a type GQ1(d18:1/18:0) species (identified only in macroalbuminuric patients) demonstrated that the present isomer is of type D (all four syalic acids are linked to the inner galactose). CONCLUSION HR MS by tandem MS methods, using an Orbitrap instrument, are a reliable tool for the identification of a particular ganglioside profile in the urine of type 2 DM patients. The sialylated species and those altered by peripheral attachments to the glycan core might be considered useful indicators of early DKD.

Gangliosides of Human Glioblastoma Multiforme: A Comprehensive Mapping and Structural Analysis by Ion Mobility Tandem Mass Spectrometry

Glioblastoma multiforme (GBM), a malignant, highly aggressive, grade IV brain tumor, which rapidly infiltrates into the nearby tissue, has drawn a significant amount of attention because of its poor prognosis and the limited treatment options available. In GBM, nearly all tumor cells exhibit aberrant cell-surface glycosylation patterns due to the alteration of their biosynthesis or postsynthesis modification process. Since gangliosides (GGs) are acknowledged as tumor-associated antigens, we have carried out here a comprehensive profiling of native ganglioside mixtures extracted and purified from GBM specimens. For this purpose, high performance ion mobility separation mass spectrometry (IMS MS) was thoroughly optimized to allow the discovery of GBM-specific structures and the assessment of their roles as tumor markers or possible associated antigens. 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, which represents 3-fold 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. By the occurrence of only one mobility feature and the diagnostic fragment ions, the IMS tandem MS conducted using collision-induced dissociation (CID) disclosed for the first time the presence of GT1c(d18:1/24:1) newly proposed here as a potential GBM marker.

Advances in the separation of gangliosides by counter-current chromatography (CCC)

Gangliosides play critical roles in the development of many progressive diseases. Due to their structural diversity, efficient methods are needed to separate individual gangliosides for studies of their functions, and for use as standards in the analysis of ganglioside mixtures. This proof-of-concept study reports a useful analytical-semi-preparative scale counter-current chromatography (CCC) enrichment of multiple ganglioside homologues of various species and classes at the milligram level.Since few individual ganglioside standards were available, this research aimed to achieve analytical-semi-preparative scale separation of gangliosides by differences in saccharide monomer compositions (classes), their arrangements (species), or ceramide compositions (homologues), using CCC. The solvent system composition, addition of solvent modifiers, and elution modes were all adjusted to separate porcine gangliosides, mainly GM1 (d36:1), GD1a (d36:1), GD1b (d36:1) and their (d38:1) homologues as a demonstration. The eluted compounds were analyzed by flow-injection analysis (FIA)-MS and LC-MS/MS.A two-phase solvent system, consisting of butanol/methyl t-butyl ether/acetonitrile/water at a ratio of 2:4:3:8 (v/v/v/v) with 0.5% (v/v) acetic acid added to the lower phase, was used to separate mg-levels of porcine gangliosides under dual-mode elution. The relative abundances of the above 6 gangliosides increased from 10 to 21% in the ganglioside extract to 55–73% in the collected fractions through the purification.

The Binding of Aβ42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation.

The binding of Aβ42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aβ42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aβ42 giving rise to more extended β-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aβ42 to sphingomyelin/cholesterol bilayers.

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

We have developed here a superior approach based on high-resolution (HR) mass spectrometry (MS) for monitoring the changes occurring with development and aging in the composition and structure of cerebellar gangliosidome. The experiments were focused on the comparative screening and structural analysis of gangliosides expressed in fetal and aged cerebellum by Orbitrap MS with nanoelectrospray ionization (nanoESI) in the negative ion mode. The employed ultrahigh-resolution MS platform allowed the discrimination, without the need of previous separation, of 159 ions corresponding to 120 distinct species in the native ganglioside mixtures from fetal and aged cerebellar biopsies, many more than detected before, when MS platforms of lower resolution were employed. A number of gangliosides, in particular polysialylated belonging to GT, GQ, GP, and GS classes, modified by O-fucosylation, O-acetylation, or CH3 COO- were discovered here, for the first time in human cerebellum. These components, found differently expressed in fetal and aged tissues, indicated that the ganglioside profile in cerebellum is development stage- and age-specific. Following the fragmentation analysis by high-energy collision-induced dissociation (HCD) tandem MS (MS/MS), we have also observed that the intimate structure of certain compounds has not changed during the development and aging of the brain, an aspect which could open new directions in the investigation of ganglioside biomarkers in cerebellar tissue.

The Structural Role of Gangliosides: Insights from X-ray Scattering on Model Membranes.

Gangliosides are an essential component of eukaryotic plasma membranes implicated in multiple physiological processes. Little is known about molecular mechanisms underlying the distribution and functions of membrane gangliosides. The overwhelmingly complex organization of glycocalyx impedes the structural analysis on cell surface and the interplay between the lipid components. Advanced X-ray analytical tools applicable to studying biological interfaces call for the simplistic models that mimic ganglioside-enriched cellular membranes. To summarize the mechanistic evidences of ganglioside interactions with lipid environment and biologically active ligands using high-resolution synchrotron X-ray scattering. A comprehensive review of studies published over the last decade was done to discuss recent accomplishments and future trends. Langmuir monolayers represent an adequate model system to assess the effect of gangliosides on membrane structure. Grazing incidence X-ray diffraction reveals a condensation effect by gangliosides on zwitterionic phospholipids with the cooperative packing of sialo- and phosphate groups. In turn, the arrangement of negatively charged lipids in ganglioside mixture remains unchanged due to the stretched conformation of carbohydrate moieties. Upon interaction with biological ligands, such as cholera toxin and galectins, the ganglioside redistribution within the ordered regions of monolayer follows distinct mechanistic patterns. The cholera toxin pentamer attached to the oligosaccharide core induces local transition from oblique to the hexagonal lattice resulting in phase coexistence. The incorporation of the A subunit responsible for endocytosis is further promoted by the acidic environment characteristic for endosomal space. X-ray reflectivity shows in-plane orientation of galectin dimers with the spatial mismatch between the lectin binding sites and ganglioside carbohydrates to perturb ceramide alkyl chains. Recent data also demonstrate sialic acid groups to be potential targets for novel peptide mimicking anticancer therapeutics. Coupled with surface X-ray scattering, the membrane mimetic approach allows for better understanding the biological role of gangliosides and their potential applications.

Electrospray ionization ion mobility mass spectrometry provides novel insights into the pattern and activity of fetal hippocampus gangliosides

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 paper, we have implemented IMS MS for the first time in the study of a highly complex native GG mixture extracted and purified from human fetal hippocampus. 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−. The detection and identification in fetal hippocampus of a much larger number of GG species than ever reported before was possible due to the ion mobility separation according to the charge state, the carbohydrate chain length and the degree of sialylation. By applying IMS in conjunction with collision induced dissociation (CID) tandem MS (MS/MS), novel GG species modified by CH3COO− attachment, 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.

Aberrant ganglioside composition in glioblastoma multiforme and peritumoral tissue: A mass spectrometry characterization

Tumor cells are characterized by aberrant glycosylation of the cell surface glycoconjugates. Gangliosides are sialylated glycosphingolipids highly abundant in neural tissue and considered as tumor markers and therapeutic targets. In this study, a detailed characterization of native ganglioside mixtures from glioblastoma multiforme, corresponding peritumoral tissue and healthy human brain was performed using mass spectrometry and high performance thin layer chromatography in order to elucidate their roles as tumor-associated antigens. Distinctive changes in ganglioside expression were determined in glioblastoma compared to healthy brain tissue showing 5 times lower total ganglioside content and higher abundance of simple gangliosides. Glioblastoma gangliosides were characterized by highly diverse ceramide composition with fatty acyl chains varying from 16 to 24 carbon atoms, while in normal and peritumoral tissue mostly C18 chains were found. The most abundant ganglioside in glioblastoma was GD3 (d18:1/18:0), followed by GD3 (d18:1/24:0) that was exclusively detected in glioblastoma tissue. Peritumoral tissue expressed higher abundance of GD3- and nLM1/GM1-species while lower GT1-species vs. normal brain. O-Ac-GD1, known as neurostatin, was detected in normal and peritumoral tissue, but not in glioblastoma. O-Ac-GD3 species were found exclusively in glioblastoma; MS structural characterization of the isomeric form possessing the O-acetylation at the inner sialic acid residue confirmed our previous finding that this isomer is glioma-associated. This, to our knowledge, the most detailed characterization of ganglioside composition in glioblastoma and peritumoral tissue, especially addressing the ceramide variability and O-acetylation of tumor-associated gangliosides, could contribute to recognition of new molecular targets for glioblastoma treatment and sub-classification.

Microfluidics-Mass Spectrometry of Protein-Carbohydrate Interactions: Applications to the Development of Therapeutics and Biomarker Discovery.

The functional interactions of carbohydrates and their protein receptors are the basis of biological events critical to the evolution of pathological states. Hence, for the past years, such interactions have become the focus of research for the development of therapeutics and discovery of novel glycan biomarkers based on their binding affinity. Due to the high sensitivity, throughput, reproducibility, and capability to ionize minor species in heterogeneous mixtures, microfluidics-mass spectrometry (MS) has recently emerged as a method of choice in protein-glycan interactomics. In this chapter, a straightforward microfluidics-based MS methodology for the assessment of protein-glycan interactions is presented. The general protocol encompasses: (1) submission of the interacting partners to a binding assay under conditions mimicking the in vivo environment; and (2) screening of the reaction products and their structural characterization by fully automated chip-nanoelectrospray (nanoESI) MS and multistage MS. The first section of the chapter is devoted to describing a method that enables the study of protein-oligosaccharide interactions by chip-nanoESI quadrupole time-of-flight (QTOF) MS and top-down complex analysis by collision-induced dissociation (CID). This section provides the protocol for the determination of the complex formed by standard β-lactoglobulin (BLG) with maltohexaose (Glc6) and recommends as a concrete application the study of the interaction between BLG extracted from human milk with Glc6, considered a ligand able to reduce the allergenicity of this protein. The second part is dedicated to presenting the protocols for the binding assay followed by chip-nanoESI ion trap (ITMS) and electron transfer dissociation (ETD) in combination with CID for protein-ganglioside interactions, using as an example the B subunit of cholera toxin (Ctb5) in interaction with comercially available GM1 species. The methodology described may be successfully applied to native ganglioside mixtures from human brain, in particular for discovery of biomarkers on the basis of their binding affinity.

B Subunit Monomers of Cholera Toxin Bind G1 Ganglioside Class as Revealed by Chip-Nanoelectrospray Multistage Mass Spectrometry

GRAPHICAL ABSTRACT

Heterogeneous Ganglioside Standards in LC-MS/MS: Sensitive Method for Quantifying the Major Molecular Components in Mono-Sialo Ganglioside Standards

A commercially-available mono-sialo (GM1) ganglioside standard consists of three major components with different ceramide structures: C18:0 fatty acid/C18-sphingosine, C18:0 fatty acid/C20-sphingosine and C20:0 fatty acid/C18-sphingosine. The usual multiple reaction monitoring (MRM) liquid chromatography-tandem mass spectrometry (LC-MS/MS) of gangliosides, which monitors the dehydrated sialic acid fragment (m/z 290), cannot differentiate between the individual iso-molecular weight components C18:0 fatty acid/C20-sphingosine and C20:0 fatty acid/C18-sphingosine. Present characterization of ganglioside standards quantifies only the fatty acid content by gas chromatography-flame ionization detection (GC-FID) analysis of the ganglioside mixture and does not parse out the percentages of the individual mono-sialo ganglioside components. In the present work analyzing a heterogeneous GM1 standard, results from a dehydrated sialic acid daughter ion MRM LC-MS/MS determination employing hydrophilic interaction liquid chromatography were combined with results of the fatty acid content determined by GC-FID analysis to sensitively quantify the three predominant individual molecular GM1 components in standards at concentrations as low as 50 ng/mL (Method 1). These dehydrated sialic acid MRM results (Method 1) were confirmed by a less sensitive fatty acid daughter ion MRM LC-MS/MS technique (Method 2) which could only determine molecular GM1 components in standards at high concentrations (1 μg/mL-10 μg/mL). Method 2, however, has the advantage of directly quantifying the three predominant individual molecular GM1 components for comparison with Method 1 results. Equations are derived which incorporate the combined data (Method 1) to calculate percentages of individual mono-sialo gangliosides in the standard. Percentages for the individual mono-sialo gangliosides in the standard differed by at most 2% (absolute difference in the percentages) in comparing the results obtained by the two methods.

Database and data analysis application for structural characterization of gangliosides and sulfated glycosphingolipids by negative ion mass spectrometry

Gangliosides and sulfated glycosphingolipids, as building and functional components of animal cell membranes, participate in cell-to-cell interactions and signaling, but also in changes of cell architecture due to different pathophysiological events. In order to enable higher throughput and to facilitate structural characterization of gangliosides/sulfo-glycosphingolipids (GSL) and their neutral GSL counterparts by negative ion mass spectrometry (MS) and tandem MS techniques, a database and data analysis application have been developed. In silico developed glycosphingolipid database considers a high diversity of ceramide compositions, several sialic acid types (N-acetylneuraminic acid, N-glycolylneuraminic acid and 2-keto-3-deoxynononic acid) as well as possible additional substitutions/modifications of glycosphingolipids, such as O-acetylation, de-N-acetylation, fucosylation, glucuronosylation, sulfation, attachment of repeating terminal hexose–N-acetylhexosamine– (Hex–HexNAc–)1–6 extension, and possible lactone forms. Data analysis application, named GSL-finder, enables correlation of negative ion MS and/or low-energy tandem MS spectra with the database structures. The GSL-database construction and the GSL-finder application searching rules are explained. Validation conducted on GD1a fraction as well as on complex mixtures of native gangliosides isolated from different mammalian brain tissues (human fetal and adult brain, and calf brain tissue) demonstrated agreement with previous studies. Plain, fast, and automated routine for structural characterization of gangliosides/sulfated glycosphingolipids and their neutral GSL counterparts described here could facilitate and improve mass spectrometric analysis of complex glycosphingolipid mixtures originating from variety of normal and pathological biomaterial, where it is known that distinctive changes in glycosphingolipid composition occur.

Nanodiscs and electrospray ionization mass spectrometry: a tool for screening glycolipids against proteins.

Electrospray ionization-mass spectrometry (ESI-MS) is extensively employed to detect and quantify protein-carbohydrate interactions in vitro and is increasingly used to screen carbohydrate libraries against target proteins. However, current ESI-MS methods are limited to carbohydrate ligands that are relatively soluble in water and are, therefore, not generally suitable for studying protein interactions with glycolipids, an important class of cellular receptors. Here, we describe a catch-and-release (CaR)-ESI-MS assay, which exploits nanodiscs (NDs) to solubilize glycolipids and mimic their natural cellular environment, suitable for screening libraries of glycosphingolipids (GSL) against proteins to identify specific interactions and to rank their relative affinities. Using the B subunit homopentamers of cholera toxin and heat labile toxin as model GSL-binding proteins, the CaR-ESI-MS was applied to NDs containing mixtures of gangliosides. The results demonstrate that the CaR-ESI-MS assay can simultaneously detect both high and low affinity GSL ligands using either a library of NDs that each contains one GSL or incorporating a mixture of GSLs into a single ND. Moreover, the relative abundances of the released ligands appear to reflect their relative affinities in solution. Application of the CaR-ESI-MS assay using NDs containing gangliosides extracted from porcine brain led to the discovery of a neolacto GSL as a cholera toxin ligand, highlighting the power of the assay for identifying specific protein-glycolipid interactions from biologically relevant mixtures.

Hydrolysis of milk gangliosides by infant-gut associated bifidobacteria determined by microfluidic chips and high-resolution mass spectrometry.

Gangliosides are receiving considerable attention because they participate in diverse biological processes. Milk gangliosides appear to block pathogen adhesion and modify the intestinal ecology of newborns. However, the interaction of milk gangliosides with gut bifidobacteria has been little investigated. The digestion products of a mixture of gangliosides isolated from milk following incubation with six strains of bifidobacteria were studied using nanoHPLC Chip Q-TOF MS. To understand ganglioside catabolism in vitro, the two major milk gangliosides--GM3 and GD3--remaining in the media after incubation with bifidobacteria were quantified. Individual gangliosides were identified through postprocessing precursor ion scans, and quantitated with the "find by molecular feature" algorithm of MassHunter Qualitative Analysis software. Bifidobacterium infantis and B. bifidum substantially degraded the GM3 and GD3, whereas B. longum subsp. longum and B. animalis subsp. lactis only showed moderate degradation. MALDI FTICR MS analysis enabled a deeper investigation of the degradation and identified ganglioside degradation specifically at the outer portions of the glycan molecules. These results indicate that certain infant gut-associated bifidobacteria have the ability to degrade milk gangliosides releasing sialic acid, and that these glycolipids could play a prebiotic role in the infant gut.

Structural analysis of brain ganglioside acetylation patterns in mice with altered ganglioside biosynthesis

Gangliosides are sialylated membrane glycosphingolipids especially abundant in mammalian brain tissue. Sialic acid O-acetylation is one of the most common structural modifications of gangliosides which considerably influences their chemical properties.In this study, gangliosides extracted from brain tissue of mice with altered ganglioside biosynthesis (St8sia1 null and B4galnt1 null mice) were structurally characterized and their acetylation pattern was analyzed. Extracted native and alkali-treated gangliosides were resolved by high performance thin layer chromatography. Ganglioside mixtures as well as separated individual ganglioside fractions were further analyzed by tandem mass spectrometry.Several O-acetylated brain ganglioside species were found in knockout mice, not present in the wild-type mice. To the best of our knowledge this is the first report on the presence of O-acetylated GD1a in St8sia1 null mice and O-acetylated GM3 species in B4galnt1 null mice. In addition, much higher diversity of abnormally accumulated brain ganglioside species regarding the structure of ceramide portion was observed in knockout versus wild-type mice.Obtained findings indicate that the diversity of brain ganglioside structures as well as acetylation patterns in mice with altered ganglioside biosynthesis, is even higher than previously reported. Further investigation is needed in order to explore the effects of acetylation on ganglioside interactions with other molecules and consequently the physiological role of acetylated ganglioside species.

Profiling and sequencing of gangliosides from human caudate nucleus by chip‐nanoelectrospray mass spectrometry

Gangliosides (GGs), sialic acid-containing glycosphingolipids are involved in many brain functions at the cell and molecular level. Compositional and structural elucidation of GGs in mixtures extracted from human brain is essential for correlating their profile with the specialized function of each brain area in health and disease. As a part of our ongoing study on GG expression and structure in different healthy and diseased brain regions, in this work, a preliminary investigation of GGs in a specimen of human caudate nucleus (CN) was carried out using an advanced mass spectrometry (MS) technique. By chip-nanoelectrospray MS performed on a NanoMate robot coupled to a high capacity ion trap instrument, 81 GG components were detected in human CN in only 1.5 min of signal acquisition. Although the native GG mixture from CN was found dominated by mono-, di- and trisialylated GGs with a slight dominance of disialylated forms (GD), four tetrasialylated structures (GQ) and two pentasialylated (GP) species were also identified. Additionally, species with unusually long fatty acid chains, exceeding 30 carbon atoms in their ceramide (Cer) composition, and several glycoforms modified by fucosyl (Fuc), O-acetyl (O-Ac) and/or lactonization were discovered. By tandem MS (MS(2) ) using collision-induced dissociation, two atypical mono and disialylated species with long-chain fatty acids in their Cer could be confirmed and structurally characterized. These results may be a starting point for new GG-based approaches in the study of CN functions and ethiopathogenesis of CN-related neurodegenerative disorders.

NEU1 and NEU3 Sialidase Activity Expressed in Human Lung Microvascular Endothelia

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

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.

High‐performance separation techniques hyphenated to mass spectrometry for ganglioside analysis

Gangliosides, sialic-acid-containing glycosphingolipids are involved in numerous biological processes and play essential roles in severe pathologies, with predilection in those of the central nervous system. Formerly, ganglioside composition and quantity were assessed exclusively by thin-layer chromatographic (TLC), immunochemical, and immunohistochemical methods, which have limited effectiveness being unable to detect minor components in mixtures of high heterogeneity. Increased awareness of the biological importance of gangliosides stimulated the development of analytical methods that are better amenable to complex ganglioside mixtures. More recently, MS in online conjunction with high-performance separation techniques brought a significant progress to the field. This review highlights the state-of-the-art development and application of separation methods online coupled to MS for ganglioside analysis. Most original and successful protocols based on GC-MS, LC-MS, and CE-MS are presented here together with the special instrumental and sample preparation requirements to be met for effective ganglioside separation, detection, and structural identification. Finally, the advantages and downsides of each methodology as well as the perspectives for simplification, standardization, and upgrading are assessed.

Application of Chip-Based Nanoelectrospray Ion Trap Mass Spectrometry to Compositional and Structural Analysis of Gangliosides in Human Fetal Cerebellum

Two native ganglioside mixtures from normal human fetal cerebellum in the 15th (Cc15) and 40th (Cc40) gestational week were subjected to NanoMate high capacity ion trap (HCT) mass spectrometric (MS) and collision induced dissociation (CID) tandem MS (MS2) analysis under thoroughly optimized experimental conditionns. An total of 56 different species were identified in Cc15 and 54 in Cc40. By employing CID MS2 molecular ions, related GD1 (d18:1/20:0) and GM2 (d18:1/19:0) species were structurally characterized in a high throughput mode. The method provided elevated ionization efficiency, high speed of analysis, almost 100% reproducibility at sample consumption per experiment situated in the femtomole range.