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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Trends in Glycolipid Biomarker Discovery in Neurodegenerative Disorders by Mass Spectrometry.

Considering the devastating effects of neurodegenerative disorders and the increasing number of people affected by them, an early diagnosis even presymptomatic, prior to serious mental deterioration is necessary. Therefore, screening for biomarkers, especially glycolipids, in the biological matrices, either tissues or body fluids has proven to be of a great help in establishing an early diagnosis of the disease.The present chapter, divided into three parts, highlights the implementation and modern applications of the most avant-garde mass spectrometry (MS) techniques characterized by speed, sensitivity and data accuracy for de novo identification and monitoring of glycolipids with potential biomarker role. The first section reviews the etiology, epidemiology, clinical picture, as well as the current diagnostic methods for four of the most frequent neurodegenerative disorders: Parkinson's disease, Alzheimer's disease, Lewy body dementia and fronto-temporal dementia. The second section is dedicated to the role of glycolipids as biomarkers of these severe conditions. In the last part of the chapter, the state of the art in terms of mass spectrometry techniques for the detection of extremely valuable glycolipid biomarkers is described in detail. The proficiency of the MS, to be considered as and further developed into a routine method for early detection of neurodegenerative disorders, is also emphasized in the chapter.

Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis.

Glycolipids (GLs), involved in biological processes and pathologies, such as viral, neurodegenerative and oncogenic transformations are in the focus of research related to method development for structural analysis. This review highlights modern separation techniques coupled to mass spectrometry (MS) for the investigation of GLs from various biological matrices. First section is dedicated to methods, which, although provide the separation in a non-liquid phase, are able to supply important data on the composition of complex mixtures. While classical thin layer chromatography (TLC) is useful for MS analyses of the fractionated samples, ultramodern ion mobility (IMS) characterized by high reproducibility facilitates to discover minor species and to apply low sample amounts, in addition to providing conformational separation with isomer discrimination. Second section highlights the advantages, applications and limitations of liquid-based separation techniques such as high performance liquid chromatography (HPLC) and hydrophilic interaction liquid chromatography (HILIC) in direct or indirect coupling to MS for glycolipidomics surveys. The on- and off-line capillary electrophoresis (CE) MS, offering a remarkable separation efficiency of GLs is also presented and critically assessed from the technical and application perspective in the final part of the review.

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. Based on ion mobility mass spectrometry (IM-MS) capability for the reliable determination of glycopatterns, the changes in the diversity and number of GGs with age and the occurrence of neurological disorders, we report here the development of a high performance IM-MS strategy for assessing the GG profile in a complex mixture extracted from a 20 year old hippocampus. IM separation of GGs based on the charge state, carbohydrate chain length and degree of sialylation led to the detection and identification of 140 species, the largest number of GGs ever reported in an adult hippocampus. Moreover, the obtained data support the concept of GG cholinergic activity. IM tandem MS experiments using collision induced dissociation (CID) confirmed the incidence of GD1b(d18:1/24:1) in the investigated hippocampus specimen.

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. From the 111 species identified, 105 were distinguished in the FL20, 74 in OL20, 46 in FL82 and 56 in OL82. The results emphasize that within the juvenile brain, GG species exhibit a higher expression in the FL than in OL, while in the aged brain the number of GG species is higher in the OL. By applying MS/MS analysis, the generated fragment ions confirmed the incidence of GT1c (d18:1/18:0) and GT1c (d18:1/20:0) in the investigated samples. The present findings are of major value for further clinical studies carried out using Orbitrap MS in order to correlate gangliosides with CNS disorders.

Electrospray Ionization Ion Mobility Mass Spectrometry of Human Brain Gangliosides.

The progress of ion mobility spectrometry (IMS), together with its association to mass spectrometry (MS), opened new directions for the identification of various metabolites in complex biological matrices. However, glycolipidomics of the human brain by IMS MS represents an area untouched up to now, because of the difficulties encountered in brain sampling, analyte extraction, and IMS MS method optimization. In this study, IMS MS was introduced in human brain ganglioside (GG) research. The efficiency of the method in clinical glycolipidomics was demonstrated on a highly complex mixture extracted from a normal fetal frontal lobe (FL37). Using this approach, a remarkably rich molecular ion pattern was discovered, which proved the presence of a large number of glycoforms and an unpredicted diversity of the ceramide chains. Moreover, the results showed for the first time the occurrence of GGs in the human brain with a much higher degree of sialylation than previously reported. Using IMS MS, the entire series starting from mono- up to octasialylated GGs was detected in FL37. These findings substantiate early clinical reports on the direct correlation between GG sialylation degree and brain developmental stage. Using IMS CID MS/MS, applied here for the first time to gangliosides, a novel, tetrasialylated O-GalNAc modified species with a potential biomarker role in brain development was structurally characterized. Under variable collision energy, a high number of sequence ions was generated for the investigated GalNAc-GQ1(d18:1/18:0) species. Several fragment ions documented the presence of the tetrasialo element attached to the inner Gal, indicating that GalNAc-GQ1(d18:1/18:0) belongs to the d series.

Applications of capillary electrophoresis electrospray ionization mass spectrometry in glycosaminoglycan analysis.

Proteoglycans (PGs) represent a class of heavily glycosylated proteins distributed in the extracellular matrix, connective tissues, and on the surface of many cell types where, as functional molecules, regulate important biological processes. Structurally, PGs consist of a core protein linked to glycosaminoglycan (GAG) chains, which basically determine the properties and activities of PGs. In view of the structural complexity of GAGs and the existing correlation between this structure and PG functions, systematic efforts are invested into development of analytical methods for GAG characterization. Although less popular and of higher technical difficulty than liquid-based chromatographic methods, CE coupled with ESI MS contributed lately an important progress to glycosaminoglycomics field. In this review article, the most significant CE ESI MS and MS/MS applications in GAG research are highlighted and critically assessed. The advantages and the limitations of each concept as well as the possible further methodological refinements are also concisely discussed. Finally, the review presents the perspectives of CE ESI MS in GAG analysis along with the objectives, which still need to be reached in the near future.

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.

Orbitrap mass spectrometry characterization of hybrid chondroitin/dermatan sulfate hexasaccharide domains expressed in brain.

In the central nervous system, chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) modulate neurotrophic effects and glial cell maturation during brain development. Previous reports revealed that GAG composition could be responsible for CS/DS activities in brain. In this work, for the structural characterization of DS- and CS-rich domains in hybrid GAG chains extracted from neural tissue, we have developed an advanced approach based on high-resolution mass spectrometry (MS) using nanoelectrospray ionization Orbitrap in the negative ion mode. Our high-resolution MS and multistage MS approach was developed and applied to hexasaccharides obtained from 4- and 14-week-old mouse brains by GAG digestion with chondroitin B and in parallel with AC I lyase. The expression of DS- and CS-rich domains in the two tissues was assessed comparatively. The analyses indicated an age-related structural variability of the CS/DS motifs. The older brain was found to contain more structures and a higher sulfation of DS-rich regions, whereas the younger brain was found to be characterized by a higher sulfation of CS-rich regions. By multistage MS using collision-induced dissociation, we also demonstrated the incidence in mouse brain of an atypical [4,5-Δ-GlcAGalNAc(IdoAGalNAc)2], presenting a bisulfated CS disaccharide formed by 3-O-sulfate-4,5-Δ-GlcA and 6-O-sulfate-GalNAc moieties.

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.

Application of ion mobility tandem mass spectrometry to compositional and structural analysis of glycopeptides extracted from the urine of a patient diagnosed with Schindler disease.

RATIONALE: Schindler disease is caused by the deficient activity of α-N-acetylgalactosaminidase, which leads to an abnormal accumulation of O-glycopeptides in tissues and body fluids. In this work the Schindler condition is for the first time approached by ion mobility (IMS) tandem mass spectrometry (MS/MS), for determining urine glycopeptide fingerprints and discriminate isomeric structures. METHODS: IMS-MS experiments were conducted on a Synapt G2s mass spectrometer operating in negative ion mode. A glycopeptide mixture extracted from the urine of a patient suffering from Schindler disease was dissolved in methanol and infused into the mass spectrometer by electrospray ionization using a syringe-pump system. MS/MS was performed by collision-induced dissociation (CID) at low energies, after mobility separation in the transfer cell. Data acquisition and processing were performed using MassLynx and Waters Driftscope software. RESULTS: IMS-MS data indicated that the attachment of one or two amino acids to the carbohydrate backbone has a minimal influence on the molecule conformation, which limits the discrimination of the free oligosaccharides from the glycosylated amino acids and dipeptides. The structural analysis by CID MS/MS in combination with IMS-MS of species exhibiting the same m/z but different configurations demonstrated for the first time the presence of positional isomers for some of the Schindler disease biomarker candidates. CONCLUSIONS: The IMS-MS and CID MS/MS platform was for the first time optimized and applied to Schindler disease glycourinome. By this approach the separation and characterization of Neu5Ac positional isomers was possible. IMS CID MS/MS showed the ability to determine the type of the glycopeptide isomers from a series of possible candidates.