Mirela Sarbu

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.

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.

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.e. 75 in Neo14 and 71 in Neo16 mixtures were identified. The ganglioside species were found characterized by a high diversity of the ceramide constitution, an elevated sialylation degree (up to pentasialylated gangliosides-GP1) and sugar cores modified by fucosylation (Fuc) and acetylation (O-Ac). Direct comparison between Neo14 and Neo16 revealed a prominent expression of monosialylated structures in the Neo16 as well as the presence of a larger number of polysialylated species in Neo14 which constitutes a clear marker of rapid development-dependant changes in the sialylation. Also the MS screening results highlighted that presumably O-acetylation process occurs faster than fucosylation. CID MS/MS under variable collision energy applied for the first time for structural analysis of a fucosylated pentasialylated species induced an efficient fragmentation with generation of ions supporting Fuc-GP1d isomer in early stage fetal brain neocortex.

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.

Automated chip-nanoelectrospray mass spectrometry for glycourinomics in Schindler disease type I.

In this study an integrative mass spectrometry (MS) approach based on fully automated chip-nanoelectrospray quadrupole time-of-flight was optimized and applied for the discovery and structural characterization of O-glycopeptides in a fraction from the urine of a patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides extracted and purified from an age matched healthy subject served as the control. 49 glycoforms were discovered in the investigated urine fraction from Schindler disease versus only 14 in control urine. Structures with relevant biological significance, previously not described, such as O-fucosylated tetrasaccharides and chains up to pentadecamers O-linked to serine, threonine, or threonine-proline were identified in the pathological urine and characterized by tandem MS (MS/MS). A number of 29 species discovered here, most of which with long chain glycans, were not previously reported as associated to this condition. All glycopeptides were detected in only 1 min analysis time, with a sample consumption situated in the femtomole range.

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.

A straightforward electrospray ionization high resolution mass spectrometry method for underivatized long chain polysaccharides

Abstract Structural analysis of long chain polysaccharides by electrospray ionization mass spectrometry (ESI-MS) is challenging since these molecules do not contain readily ionizable groups. Their mass spectra are dominated by singly charged ions, limiting the detection of high molecular weight species. Derivatization can enhance ionization, but analyte loss on purification decreases sensitivity. We report a method based on nanoESI-MS and MS/MS by collision induced dissociation (CID) for underivatized long chain polysaccharides. The procedure was tested on underivatized polydisperse dextrans (average molecular weight 4,000) at 2.6 kV ESI voltage and CID MS/MS at energies between 30-60 eV. 113 ions corresponding to species from Glc2 to Glc35 were detected. Ions at m/z 1,409.48, 1,107.35 and 1,438.47, assigned to [G17+2Na]2+,[G20+H+Na+K]3+ and [G35+2H+Na+K]4+, were sequenced and characterized by MS/MS. The component containing 35 Glc repeats is the longest polysaccharide chain detected by ESI-MS and structurally analyzed by MS/MS without prior derivatization and/or separation. Graphical Abstract

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.

Structural analysis by electrospray ionization mass spectrometry of GT1 ganglioside fraction isolated from fetal brain

GRAPHICAL ABSTRACT ABSTRACT Due to the structural complexity of gangliosides and the lack of data about the minor but still biologically relevant species, in the present study, a trisialoganglioside (GT1) fraction isolated from a 27th gestational week fetal brain was investigated using high resolution (HR) mass spectrometry (MS) with sample infusion by chip nanoelectrospray. The data provided reliable insights into the polysialylated ganglioside expression and its structure, enlarging the inventory of the identified fetal brain glycoforms. Moreover, 18 new GT1 species were for the first time detected and characterized, using the high performance analytical platform encompassing fractionation, chip-based ionization, and HR and tandem MS.

Nanofluidics-Based Mass Spectrometry. Applications for Biomarker Discovery in Lysosomal Storage Diseases

Substantial efforts are currently invested in development of micro- and nanofluidics-based systems as front end technology for electrospray ionization (ESI) mass spectrometry (MS). Since its first introduction in biological MS chip-based ESI demonstrated a high potential to discover novel biopolymer species due to the efficient ionization properties, preferential formation of multiply charged ions and the elevated reproducibility and sensitivity. In combination with high-resolution mass spectrometers or instruments able to perform multistage fragmentation, chip-electrospray confirmed its unique ability to offer structural elucidation of minor species in complex mixtures, which often represent valuable biomarkers of severe diseases. This aspect is of particular importance for the applicability of chip MS in clinical investigation where only minute amounts of biological material are available. In view of these major advantages of nanofluidics in conjunction with modern MS, this chapter reviews the strategies, which allowed a successful application of chip technology for early diagnostic of lysosomal storage diseases. The first part is dedicated to the principles of ESI MS and to advanced nanochip systems for ESI MS. The second part highlights important achievements of nanochip ESI MS in biomarker discovery and diagnostic of LSDs such as Fabry and Schindler diseases. Finally, this chapter emphasizes that advanced chip ESI MS has real perspectives to become a routine method for early diagnosis and therapy of severe pathologies such as LSDs.