Human Cerebellum Gangliosides: A Comprehensive Analysis by Ion Mobility Tandem Mass Spectrometry.

The human cerebellum is an ultraspecialized region of the brain responsible for cognitive functions and movement coordination. The fine mechanisms through which the process of aging impacts such functions are not well understood; therefore, a rigorous exploration of this brain region at the molecular level is deemed necessary. Gangliosides, sialylated glycosphingolipids, highly and specifically expressed in the human central nervous system, represent possible molecular markers of cerebellum development and aging.

Advances in Mass Spectrometry of Gangliosides Expressed in Brain Cancers.

Gangliosides are highly abundant in the human brain where they are involved in major biological events. In brain cancers, alterations of ganglioside pattern occur, some of which being correlated with neoplastic transformation, while others with tumor proliferation. Of all techniques, mass spectrometry (MS) has proven to be one of the most effective in gangliosidomics, due to its ability to characterize heterogeneous mixtures and discover species with biomarker value.

Ion Mobility Mass Spectrometry Reveals Rare Sialylated Glycosphingolipid Structures in Human Cerebrospinal Fluid.

Gangliosides (GGs) represent an important class of biomolecules associated with the central nervous system (CNS). In view of their special role at a CNS level, GGs are valuable diagnostic markers and prospective therapeutic agents. By ion mobility separation mass spectrometry (IMS MS), recently implemented by us in the investigation of human CNS gangliosidome, we previously discovered a similarity between GG profiles in CSF and the brain.

Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts.

The recent identification of plasma membrane (Ca)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains.

High-resolution mass spectrometry reveals a complex ganglioside pattern and novel polysialylated structures associated with the human motor cortex.

We have developed here a superior methodology based on high-resolution mass spectrometry for screening and fragmentation analysis of gangliosides extracted and purified from the human motor cortex . The experiments, conducted on a nanoelectrospray Orbitrap mass spectroscope in the negative ion mode, allowed the discrimination in the native mixture extracted from human motor cortex of no less than 83 different gangliosides, which represents the highest number of structures identified so far in this brain region. The spectral data, acquired in high-resolution mass spectrometry mode with a remarkable sensitivity and an average mass accuracy of 4.

Ganglioside Composition Distinguishes Anaplastic Ganglioglioma Tumor Tissue from Peritumoral Brain Tissue: Complementary Mass Spectrometry and Thin-Layer Chromatography Evidence.

Gangliosides serve as antitumor therapy targets and aberrations in their composition strongly correlate with tumor growth and invasiveness. Anaplastic ganglioglioma is a rare, poorly characterized, malignant neuronal-glial tumor type. We present the first comparative characterization of ganglioside composition in anaplastic ganglioglioma vs.

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.

High resolution mass spectrometry provides novel insights into the ganglioside pattern of brain cavernous hemangioma.

In this study we have optimized nanoelectrospray ionization (nanoESI) high resolution mass spectrometry (HR MS) performed on Orbitrap instrument in the negative ion mode for the determination of the composition and structure of gangliosides extracted from human brain cavernous hemangioma. The optimized HR MS platform, allowed the discrimination of 62 ions, corresponding to 52 different ganglioside species, which represents roughly twice the number of species existing in the current inventory of human brain hemangioma-associated gangliosides. The experiments revealed a ganglioside pattern dominated by GD-type of structures as well as an elevated incidence of species characterized by a low degree of sialylation and short glycan chains, including asialo GA1 (d18:1/18:0), which offer a new perspective upon the ganglioside composition in this benign tumor.

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.

Cerebrospinal fluid: Profiling and fragmentation of gangliosides by ion mobility mass spectrometry.

The proximity of cerebrospinal fluid (CSF) with the brain, its permanent renewal and better availability for research than tissue biopsies, as well as ganglioside (GG) shedding from brain to CSF, impelled lately the development of protocols for the characterization of these glycoconjugates and discovery of central nervous system biomarkers expressed in CSF. Currently, the investigation of CSF gangliosides is focused on concentration measurements of the predominant classes and much less on their profiling and structural analysis. Since we have demonstrated recently the high performance of ion mobility separation mass spectrometry (IMS MS) for compositional and structural determination of human brain GGs, in the present study we have implemented for the first time IMS MS for the exploration of human CSF gangliosidome, in order to generate the first robust mass spectral database of CSF gangliosides.

Gangliosidome of human anencephaly: A high resolution multistage mass spectrometry study.

Widely dispersed throughout the entire body tissues, gangliosides (GGs) are essential components of neuronal cell membranes, where exhibit a vital role in neuronal function and brain development, directly influencing the neural tube formation, neurogenesis, neurotransmission, etc. Due to several factors, partial or complete closing faults of the fetal neural tube may occur in the first trimester of pregnancy, generating a series of neural tube defects (NTD), among which anencephaly. The absence in anencephaly of the forebrain and skull bones determines the exposure to the amniotic fluid of the remaining brain tissue and the spinal cord, causing the degeneration of the nervous system tissue.

Ion mobility mass spectrometry provides novel insights into the expression and structure of gangliosides in the normal adult human hippocampus.

Clustered into the so-called "glycosynaptic" microdomains in the central nervous system (CNS), gangliosides (GGs) are involved in the formation of functional synapses and neural circuits. Therefore, GGs are important biomarkers in the early diagnosis of CNS pathologies, which are the focus of our research as potential therapeutic targets. A series of neuropsychiatric disorders, including Alzheimer's disease and schizophrenia, are characterized by amnesia and disorientation caused by hippocampal atrophy and diminished cholinergic activity.

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.

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.

Assessment of ganglioside age-related and topographic specificity in human brain by Orbitrap mass spectrometry.

The gangliosides (GGs) of the central nervous system (CNS) exhibit age and topographic specificity and these patterns may correlate with the functions and pathologies of the brain regions. Here, chloroform extraction, nanoelectrospray (nanoESI) negative ionization, together with Orbitrap high resolution mass spectrometry (MS) determined the topographic and age-related GG specificity in normal adult human brain. Mapping of GG mixtures extracted from 20 to 82 year old frontal and occipital lobes revealed besides a decrease in the GG number with age, a variability of sialylation degree within the brain regions.

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.

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.

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.

Early stage fetal neocortex exhibits a complex ganglioside profile as revealed by high resolution tandem mass spectrometry.

In this study we report on the first mass spectrometric (MS) investigation of gangliosides and preliminary assessment of the expression and structure in normal fetal neocortex in early developmental stages: 14th (Neo14) and 16th (Neo16) gestational weeks. Ganglioside analysis was carried out using a hybrid quadrupole time-of-flight (QTOF) MS with direct sample infusion by nanoelectrospray ionization (nanoESI) in the negative ion mode. Under optimized conditions a large number of glycoforms i.

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.

Profiling and sequence analysis of gangliosides in human astrocytoma by high-resolution mass spectrometry.

In this preliminary investigation, a low-grade astrocytoma (AcT) is investigated by high-resolution (HR) mass spectrometry (MS) aiming at characterization of gangliosides with potential biomarker value. The research was conducted towards a comparative mapping of ganglioside expression in AcT, its surrounding tissue (ST) and a normal control brain tissue (NT). HR MS was conducted in the negative ion mode nanoelectrospray ionization (nanoESI).

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.

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.

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.

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.