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. In this context, for a comprehensive determination of development- and age-specific components, we have conducted here a comparative profiling and structural determination of the gangliosides expressed in fetal cerebellum in two intrauterine developmental stages and aged cerebellum by ion mobility separation (IMS) mass spectrometry (MS) and tandem MS (MS/MS). Due to the high sensitivity and efficiency of separation provided by IMS MS, no less than 551 chemically distinct species were identified, which represents 4.5 times more gangliosides than ever discovered in this brain region. The detailed assessment of fetal vs aged cerebellum gangliosidome showed marked discrepancies not only in the general number of the species expressed, but also in their sialylation patterns, the modifications of the glycan core, and the composition of the ceramides. All of these characteristics are potential markers of cerebellum development and aging. The structural analysis by collision-induced dissociation (CID) documented the occurrence of GD1b (d18:1/18:0) isomer in the fetal cerebellum in the second gestational trimester, with all probability of GQ1b (t18:1/18:0) in the near-term fetus and of GQ1b (d18:1/18:0) in aged cerebellum.

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. This review highlights the most significant achievements of MS in the analysis of gangliosides in human brain cancers. The first part presents the latest state of MS development in the discovery of ganglioside markers in primary brain tumors, with a particular emphasis on the ion mobility separation (IMS) MS and its contribution to the elucidation of the gangliosidome associated with aggressive tumors. The second part is focused on MS of gangliosides in brain metastases, highlighting the ability of matrix-assisted laser desorption/ionization (MALDI)-MS, microfluidics-MS and tandem MS to decipher and structurally characterize species involved in the metastatic process. In the end, several conclusions and perspectives are presented, among which the need for development of reliable software and a user-friendly structural database as a search platform in brain tumor diagnostics.

Glycomics by ion mobility tandem mass spectrometry of chondroitin sulfate disaccharide domain in biglycan.

Biglycan (BGN), a small leucine-rich repeat proteoglycan, is involved in a variety of pathological processes including malignant transformation, for which the upregulation of BGN was found related to cancer cell invasiveness. Because the functions of BGN are mediated by its chondroitin/dermatan sulfate (CS/DS) chains through the sulfates, the determination of CS/DS structure and sulfation pattern is of major importance. In this study, we have implemented an advanced glycomics method based on ion mobility separation (IMS) mass spectrometry (MS) and tandem MS (MS/MS) to characterize the CS disaccharide domains in BGN. The high separation efficiency and sensitivity of this technique allowed the discrimination of five distinct CS disaccharide motifs, of which four irregulated in their sulfation pattern. For the first time, trisulfated unsaturated and bisulfated saturated disaccharides were found in BGN, the latter species documenting the non-reducing end of the chains. The structural investigation by IMS MS/MS disclosed that in one or both of the CS/DS chains, the non-reducing end is 3-O-sulfated GlcA in a rather rare bisulfated motif having the structure 3-O-sulfated GlcA-4-O-sulfated GalNAc. Considering the role played by BGN in cancer cell spreading, the influence on this process of the newly identified sequences will be investigated in the future.

Ganglioside analysis in body fluids by liquid-phase separation techniques hyphenated to mass spectrometry.

The expression of gangliosides in central nervous system is a few times higher than in the extraneural tissue, a characteristic highlighting their major role at this level. Although in very low amounts, gangliosides are ubiquitously distributed in body fluids too, where, depending on many factors, including pathological states, their composition fluctuates, thus having diagnostic value. Ganglioside investigation in biological fluids, which, except for cerebrospinal fluid (CSF), may be sampled noninvasively, was for years impeded by the limited sensitivity of the analytical instrumentation available in glycomics. However, because the last decade has witnessed significant developments in biological mass spectrometry (MS) and the hyphenated separation techniques, marked by a major increase in sensitivity, reproducibility, and data reliability, ganglioside research started to be focused on biofluid analysis by separation techniques coupled to MS. In this context, our review presents the achievements in this emerging field of gangliosidomics, with a particular emphasis on modern liquid chromatography (LC), thin-layer chromatography, hydrophilic interaction LC, and ion mobility separation coupled to high-performance MS, as well as the results generated by these systems and allied experimental procedures in profiling and structural analysis of gangliosides in healthy or diseased body fluids, such as CSF, plasma/serum, and milk.

Structural Investigation of Betulinic Acid Plasma Metabolites by Tandem Mass Spectrometry.

Betulinic acid (BA) has been extensively studied in recent years mainly for its antiproliferative and antitumor effect in various types of cancers. Limited data are available regarding the pharmacokinetic profile of BA, particularly its metabolic transformation in vivo. In this study, we present the screening and structural investigations by ESI Orbitrap MS in the negative ion mode and CID MS/MS of phase I and phase II metabolites detected in mouse plasma after the intraperitoneal administration of a nanoemulsion containing BA in SKH 1 female mice. Obtained results indicate that the main phase I metabolic reactions that BA undergoes are monohydroxylation, dihydroxylation, oxidation and hydrogenation, while phase II reactions involved sulfation, glucuronidation and methylation. The fragmentation pathway for BA and its plasma metabolites were elucidated by sequencing of the precursor ions by CID MS MS experiments.

Identification and Structural Characterization of Novel Chondroitin/Dermatan Sulfate Hexassacharide Domains in Human Decorin by Ion Mobility Tandem Mass Spectrometry.

Chondroitin sulfate (CS) and dermatan sulfate (DS) are found in nature linked to proteoglycans, most often as hybrid CS/DS chains. In the extracellular matrix, where they are highly expressed, CS/DS are involved in fundamental processes and various pathologies. The structural diversity of CS/DS domains gave rise to efforts for the development of efficient analytical methods, among which is mass spectrometry (MS), one of the most resourceful techniques for the identification of novel species and their structure elucidation. In this context, we report here on the introduction of a fast, sensitive, and reliable approach based on ion mobility separation (IMS) MS and MS/MS by collision-induced dissociation (CID), for the profiling and structural analysis of CS/DS hexasaccharide domains in human embryonic kidney HEK293 cells decorin (DCN), obtained after CS/DS chain releasing by ß-elimination, depolymerization using chondroitin AC I lyase, and fractionation by size-exclusion chromatography. By IMS MS, we were able to find novel CS/DS species, i.e., under- and oversulfated hexasaccharide domains in the released CS/DS chain. In the last stage of analysis, the optimized IMS CID MS/MS provided a series of diagnostic fragment ions crucial for the characterization of the misregulations, which occurred in the sulfation code of the trisulfated-4,5-Δ-GlcAGalNAc[IdoAGalNAc]2 sequence, due to the unusual sulfation sites.

Experimental and Computational Study of Novel Pyrazole Azo Dyes as Colored Materials for Light Color Paints.

This paper presents the synthesis of eight new pyrazole azo dyes using ethyl 5-amino-3-methyl-1H-pyrazole-4-carboxylate as the diazotization component and various active methylene derivatives as coupling components. These new azo dyes were characterized by spectroscopic (FT-IR, UV-VIS), and spectrometric (1H NMR, 13C NMR, MS) analyses. The dye structures were modeled by the MMFF94s force field and quantum chemical density functional theory (DFT) calculations using the B3LYP functional and the 6-311G(d,p) basis set, in the gas phase. Weak electrostatic hydrogen bonds for the azo and hydrazo dye tautomers were found in the ground state. The CIS, TD (using the B3LYP and M06-2X functionals), and ZINDO methods were used to estimate the dye UV-VIS spectra in ethanol, which were compared with the experimental ones. The anti-configuration arrangement of the π-bonds and the presence of the prevalent hydrazo dye tautomer were supported by the computed 1H NMR and 13C NMR spectra. A good accordance between the experimental and predicted absorption maxima and chemical shifts was observed. Color investigations using the CIEL*a*b* space were conducted for all dyes in powder and for their mixtures in water-based acrylic resins. The results confirm the newly synthesized dyes' color properties and that they might be used for light color paints in the varnishes industry.

Capillary Zone Electrophoresis-Electrospray Ionization Tandem Mass Spectrometry for Total Analysis of Chondroitin/Dermatan Sulfate Oligosaccharides.

Proteoglycans are heavily glycosylated proteins, covalently linked to one or more glycosaminoglycan (GAG) chains, abundantly expressed in the extracellular matrix (ECM). Among GAGs, chondroitin sulfate (CS) and dermatan sulfate (DS) play an essential role at the ECM level; however, the composition of the hybrid CS/DS as well as the distribution of the sulfate groups along the chain were also shown to influence biological activities in brain. The elevated structural diversity of CS/DS motifs, in which sulfation may occur at GalNAc and/or IdoA/GlcA in various combinations, requires the development of specific high performance analytical methods for reliable elucidation. Due to its sensitivity, reproducibility, and efficiency, capillary zone electrophoresis (CZE) for separation of CS/DS oligosaccharides coupled to electrospray ionization mass spectrometry (ESI-MS) for their structure determination contributed an essential progress to this field.In the present chapter, two powerful methods based on CZE for separation and ESI-MS for identification and structural analysis of CS/DS are presented. The first part is devoted to offline CZE-ESI-MS based on fraction collection, screening by negative ion mode nanoESI, and fragmentation analysis in tandem MS using collision-induced dissociation (CID) at low ion acceleration energies. In the second part of the chapter, a strategy for online CZE-ESI-MS in normal polarity and negative mode ESI followed by tandem MS in real-time data-dependent acquisition mode for CS/DS separation, screening, and fragmentation is described in detail. The latter method entails the in-laboratory manufacturing of a simple yet sturdy interface for the online CZE coupling to ESI-MS and the optimization of the coupled system for total analysis of regularly sulfated and irregularly, i.e., under- and oversulfated CS/DS domains.

Gangliosidome of a Human Hippocampus in Temporal Lobe Epilepsy Resolved by High-Resolution Tandem Mass Spectrometry.

In this study, we developed a high-resolution tandem mass spectrometry (HR MS) approach to assess presumed changes in gangliosidome of a human hippocampus affected by temporal lobe epilepsy (TLE) in comparison with a normal hippocampus. Gangliosides, membrane glycolipids, are particularly diverse and abundant in the human brain, and participate in ion transport and modulation of neuronal excitability. Changes in structural ganglioside pattern potentially linked to TLE molecular pathogenesis have not been explored in detail. Aiming to characterize TLE-specific gangliosidome, we analyzed the native gangliosides purified from a human hippocampal tissue sample affected by TLE and a control hippocampus using HR MS. Marked differences of ganglioside expression were shown in TLE vs. control, particularly with respect to the sialylation degree of components, discovered as a characteristic feature of TLE. Another major finding is the occurrence of tetrasialofucogangliosides in TLE and species modified by either O-acetylation or CH3COO-. Structural analysis by higher-energy collisional dissociation (HCD) MS/MS gave rise to fragmentation patterns implying that the GQ1b (d18:1/18:0) isomer is specifically associated with TLE. Further investigation in a larger sample is needed in order to confirm the discovery of ganglioside structures specifically expressed in human TLE and to provide information on the probable role of gangliosides in the molecular events underlying seizures.

High-Resolution Tandem Mass Spectrometry Identifies a Particular Ganglioside Pattern in Early Diabetic Kidney Disease of Type 2 Diabetes Mellitus Patients.

Considering the valuable information provided by glycosphingolipids as molecular markers and the limited data available for their detection and characterization in patients suffering from Type 2 diabetic kidney disease (DKD), we developed and implemented a superior method based on high-resolution (HR) mass spectrometry (MS) and tandem MS (MS/MS) for the determination of gangliosides in the urine of DKD patients. This study was focused on: (i) testing of the HR MS and MS/MS feasibility and performances in mapping and sequencing of renal gangliosides in Type 2 DM patients; (ii) determination of the changes in the urine gangliosidome of DKD patients in different stages of the disease-normo-, micro-, and macroalbuminuria-in a comparative assay with healthy controls. Due to the high resolution and mass accuracy, the comparative MS screening revealed that the sialylation status of the ganglioside components; their modification by O-acetyl, CH3COO-, O-fucosyl, and O-GalNAc; as well as the composition of the ceramide represent possible markers for early DKD detection, the assessment of disease progression, and follow-up treatment. Moreover, structural investigation by MS/MS demonstrated that GQ1d(d18:1/18:0), GT1α(d18:1/18:0) and GT1b(d18:1/18:0) isomers are associated with macroalbuminuria, meriting further investigation in relation to their role in DKD.

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. Based on these findings, we developed IMS tandem MS (MS/MS) to characterize rare human CSF glycoforms, with a potential biomarker role. To investigate the oligosaccharide and ceramide structures, the ions detected following IMS MS separation were submitted to structural analysis by collision-induced dissociation (CID) MS/MS in the transfer cell. The IMS evidence on only one mobility feature, together with the diagnostic fragment ions, allowed the unequivocal identification of isomers in the CSF. Hence, by IMS MS/MS, GalNAc-GD1c(d18:1/18:1) and GalNAc-GD1c(d18:1/18:0) having both Neu5Ac residues and GalNAc attached to the external galactose were for the first time discovered and structurally characterized. The present results demonstrate the high potential of IMS MS/MS for biomarker discovery and characterization in body fluids, and the perspectives of method implementation in clinical analyses targeting the early diagnosis of CNS diseases through molecular fingerprints.

Gangliosides as Biomarkers of Human Brain Diseases: Trends in Discovery and Characterization by High-Performance Mass Spectrometry.

Gangliosides are effective biochemical markers of brain pathologies, being also in the focus of research as potential therapeutic targets. Accurate brain ganglioside mapping is an essential requirement for correlating the specificity of their composition with a certain pathological state and establishing a well-defined set of biomarkers. Among all bioanalytical methods conceived for this purpose, mass spectrometry (MS) has developed into one of the most valuable, due to the wealth and consistency of structural information provided. In this context, the present article reviews the achievements of MS in discovery and structural analysis of gangliosides associated with severe brain pathologies. The first part is dedicated to the contributions of MS in the assessment of ganglioside composition and role in the specific neurodegenerative disorders: Alzheimer's and Parkinson's diseases. A large subsequent section is devoted to cephalic disorders (CD), with an emphasis on the MS of gangliosides in anencephaly, the most common and severe disease in the CD spectrum. The last part is focused on the major accomplishments of MS-based methods in the discovery of ganglioside species, which are associated with primary and secondary brain tumors and may either facilitate an early diagnosis or represent target molecules for immunotherapy oriented against brain cancers.

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 4.48 ppm, also show that the gangliosidome of motor cortex is generally characterized by species exhibiting a much higher degree of sialylation than previously known. Motor cortex was found dominated by complex structures with a sialylation degree ≥3, exhibiting long saccharide chains, in the G1 class. Fucogangliosides and species with the glycan chain elongated by either O-acetylation and/or acetate anion attachments were also detected; the later modification was for the first time discovered in this brain region. Of major significance is the identification of hepta and octasialylated species of GS1 and GO1 type, which are among the structures with the longest oligosaccharide chain discovered so far in the human brain. In the last stage of research, tandem mass spectrometry performed by higher energy collision dissociation provided structural data documenting the occurrence of GT1b (d18:1/20:0) isomer in the human motor cortex.

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.

Developments and applications of separation and microfluidics methods coupled to electrospray mass spectrometry in glycomics of nervous system gangliosides.

Gangliosides are particularly abundant in the nervous system (NS) where their pattern and structure in a certain milieu or a defined region exhibit a pronounced specificity. Since gangliosides are useful biomarkers for diagnosis of NS ailments, a clear-cut mapping of individual components represents a prerequisite for designing ganglioside-based diagnostic procedures, treatments, or vaccines. These bioclinical aspects and the high diversity of ganglioside species claim for development of specific analytical strategies. This review summarizes the state-of-the-art in the implementation of separation techniques and microfluidics coupled to MS, which have contributed significantly to the advancement of the field. In the first part, the review discusses relevant approaches based on HPLC MS and CE coupled to ESI MS and their applications in the characterization of gangliosides expressed in healthy and diseased NS. A considerable section is dedicated to microfluidics MS and ion mobility separation MS, developed for the study of brain gangliosidome and its changes triggered by various factors, as well as for ganglioside biomarker discovery in neurodegenerative diseases and brain cancer. In the last part of the review, the benefits and perspectives in ganglioside research of these high-performance techniques are presented.

Urinary proteins detected using modern proteomics intervene in early type 2 diabetic kidney disease - a pilot study.

Aim: An advanced proteomics platform for protein biomarker discovery in diabetic chronic kidney disease (DKD) was developed, validated and implemented. Materials & methods: Three Type 2 diabetes mellitus patients and three control subjects were enrolled. Urinary peptides were extracted, samples were analyzed on a hybrid LTQ-Orbitrap Velos Pro instrument. Raw data were searched using the SEQUEST algorithm and integrated into Proteome Discoverer platform. Results & discussion: Unique peptide sequences, resulted sequence coverage, scoring of peptide spectrum matches were reported to albuminuria and databases. Five proteins that can be associated with early DKD were found: apolipoprotein AI, neutrophil gelatinase-associated lipocalin, cytidine deaminase, S100-A8 and hemoglobin subunit delta. Conclusion: Urinary proteome analysis could be used to evaluate mechanisms of pathogenesis of DKD.

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. Many of the structures are characteristic for this type of tumor only and are to be considered in further investigations for their potential use in early brain hemangioma diagnosis based on molecular markers. The detailed fragmentation analysis performed by collision-induced dissociation (CID) tandem MS provided information of structural elements related to the glycan core and ceramide moiety, which confirmed the molecular configuration of GD3 (d18:1/24:1) and GD3 (d18:1/24:2) species with potential biomarker role.

Ion mobility mass spectrometry of human melanoma gangliosides.

Malignant melanoma is an aggressive type of skin cancer, rarely detected in the early stages. Various sets of methods and techniques, including dermatoscopical inspection of the "ABCDE" signs of the lesion, imaging techniques or microscopical, immunohistochemical and serological biomarkers are available and used nowadays to diagnose malignant melanoma. To date, different biomarkers were proposed for melanoma, but only a few, including circulating proteins, such as lactate dehydrogenase, molecular and metabolite biomarkers, have reached clinical applications. Gangliosides represent an emerging class, being used as tumor markers and targets of antibody therapy in melanomas, based on their elevated abundance in melanoma, especially of GM3 and GD3, when compared with the corresponding normal tissues. The conjunction of mass spectrometry (MS) with ion mobility separation (IMS) demonstrated an elevated potential in detection and identification of low abundant components, with biomarker role, in extremely complex biological mixtures. Therefore, here, a native ganglioside extract originating from human melanoma was investigated for the first time by IMS MS to provide the first profiling of gangliosides in this type of cancer. The present approach revealed the high incidence of species belonging to GD3 and GM3 classes, as well as of de-N-acetyl GM3 (d-GM3) and de-N-acetyl GD3 (d-GD3), characteristic for human melanoma. Additionally, the structure of two molecules characterized by shorter glycan chains associated to melanoma, were investigated in detail. The present approach brings valuable data related to this type of cancer, completing the existing inventory of melanoma-associated biomarkers and opens new directions for further research in this field.

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.

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. IMS MS separation and screening revealed 113 distinct GG species in CSF, having similar compositions to the species detected in human brain. In comparison with the brain tissue, we have discovered in CSF several components containing fatty acids with odd number of carbon atoms and/or short glycan chains. By tandem MS (MS/MS) we have further analyzed the structure of GD3(d18:1/18:0) and GD2(d18:1/18:0), two glycoforms exhibiting short carbohydrate chains found in CSF, but discovered and characterized previously in brain as well. According to the present results, human CSF and brain show a similar ganglioside pattern, a finding that might be useful in clinical research focused on discovery of ganglioside species associated to neurodegenerative diseases and brain tumors.