Željka Vukelić

Coupling of fully automated chip-based electrospray ionization to high-capacity ion trap mass spectrometer for ganglioside analysis

NanoMate robot was coupled to a high-capacity ion trap (HCT) mass spectrometer to create a system merging automatic chip-based electrospray ionization (ESI) infusion, ultrafast ion detection, and multistage sequencing at superior sensitivity. The interface between the NanoMate and HCT mass spectrometer consists of an in-laboratory constructed mounting device that allows adjustment of the robot position with respect to the mass spectrometer inlet. The coupling was optimized for ganglioside (GG) high-throughput analysis in the negative ion mode and was implemented in clinical glycolipidomics for identification and structural characterization of anencephaly-associated species. By NanoMate HCT mass spectrometry (MS), data corroborating significant differences in GG expression in anencephalic versus age-matched normal brain tissue were collected. The feasibility of chip-based nanoESI HCT multistage collision-induced dissociation (CID MS(n)) for polysialylated GG fragmentation and isomer discrimination was tested on a GT1 (d18:1/18:0) anencephaly-associated structure. MS(2)-MS(4) obtained by accumulating scans at variable fragmentation amplitudes gave rise to the first fragmentation patterns from which the presence of GT1b structural isomer could be determined unequivocally without the need for supplementary investigation by any other analytical or biochemical methods.

A capillary electrophoresis and off-line capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry approach for ganglioside analysis

A systematic study for the optimization and implementation of high‐performance capillary electrophoresis (HPCE) in conjunction with negative ion electrospray ionization‐quadrupole time of flight‐tandem mass spectrometry (ESI‐QTOF‐MS/MS) for the analysis of complex glycolipids is described. The performance of the capillary electrophoresis (CE) and off‐line CE/ESI‐QTOF‐MS approach has been explored for screening a complex ganglioside mixture from bovine brain. All instrumental and solution parameters demonstrated to require special adjustment and to have the most substantial effect on the CE separation, abundance of product ions produced in a low‐energy collision‐induced dissociation (CID) process and their detection by MS/MS, when attempting to identify and sequence single ganglioside molecular species from CE eluted fractions. Upon optimization of the experimental parameters, an efficient methodology emerged providing the general basic requirements for combined CE/ESI‐MS analysis of this type of complex glycoconjugate.

Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry

AbstractWe 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. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS2 mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor. Human brain biomarker discovery by advanced chipbased nanoelectrospray mass spectrometry

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. In this work, for the first time, a high-throughput mapping and sequencing of gangliosides in human fetal brain was performed by a novel mass spectrometry (MS)-based approach developed recently in our laboratory. Three GG mixtures extracted and purified from different regions of the same fetal brain in the 36th gestational week: frontal neocortex (NEO36), white matter of the frontal lobe (FL36) and white matter of the occipital lobe (OL36) were subjected to comparative high-throughput screening and multi-stage fragmentation by fully automated chip-based nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT) MS. Using this method, in only a few minutes of signal acquisitions, over 100 GG and asialo-GG species were detected and identified in the three mixtures. Obtained data revealed for the first time that differences in GG expression in human fetal brain are dependent on phylogenetic development rather than topographic factors. While a significant variation of GG distribution in NEO36 vs FL36 was observed, no significant differences in GG expression in white matter of frontal vs occipital lobe were detected. Additionally, the largest number of species was identified in NEO36, which correlates with the functional complexity of neocortex as the newest brain region. In the last stage of analysis, using MS2–MS3 molecular ion fragmentation at variable amplitudes, a NEO36-associated GD1b isomer could clearly be discriminated. Present results indicate that the combination of fully automated chipESI with HCT MS n is able to provide ultra-fast, sensitive and reliable analyses of complex lipid-linked carbohydrates from which the pattern of their expression and structure in a certain type of bio-matrix can be determined.

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

AbstractIn 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). Fragmentation analysis was carried out by collision-induced dissociation (CID) MS2–MS4. Due to the high resolving power and mass accuracy, by comparative mapping of the ganglioside extracts from AcT, ST and NT, under identical conditions, 37 different species in AcT, 40 in ST and 56 in NT were identified. AcT and ST were found to contain 18 identical ganglioside components. Among all three specimens, ST extract presented the highest levels of sialylation, fucosylation and acetylation, a feature which might be correlated to the tumor expansion in the adjacent brain area. MS mapping indicated also that AcT, ST and NT share one doubly deprotonated molecule at m/z 1063.31, attributable to GT1(d18:1/18:0) or GT1(d18:0/18:1). CID MS2–MS4 on these particular ions detected in AcT and ST provided data supporting GT1c isomer in the investigated astrocytoma tissue. Our results show that HR MS has a remarkable potential in brain cancer research for the determination of tumor-associated markers and for their structural determination. FigureGanglioside isomer discrimination in human astrocytoma by Orbitrap multistage MS

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. All structures presented long‐chain bases with 18 carbon atoms, while the length of the fatty acid was found to vary from C11 to C25. MS screening results indicated also that the diversity of the expressed GM1 structures is higher than expected in view of the low proportions evidenced by densitometric quantification. Simultaneous fragmentation of meningioma‐associated GM1 (d18:1/24:1) and GM1 (d18:1/24:0) by MS/MS using CID confirmed the postulated structures of the ceramide moieties and provided data on the glycan core, which document that for each of the GM1 (d18:1/24:1) and GM1 (d18:1/24:0) forms both GM1a and GM1b isomers are expressed in the investigated meningioma tissue.

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.

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

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

APPLICATION OF CHIP-BASED NANOELECTROSPRAY ION TRAP MASS SPECTROMETRY TO COMPOSITIONAL AND STRUCTURAL ANALYSIS OF GANGLIOSIDES IN HUMAN FETAL CEREBELLUM

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

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.