Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.     

Gangliosides from human granulocytes: a nano-ESI QTOF mass spectrometry fucosylation study of low abundance species in complex mixtures

Nano-ESI QTOF MS was used for sensitive mapping and sequencing of single molecular species in complex ganglioside mixtures obtained from human granulocytes, where the fucosylated carbohydrate chains of granulocyte gangliosides carry sLex and VIM-2 epitopes postulated to interact with E-selectin of the blood vessel wall in the early phase of the inflammation process. Functionally relevant components are expressed only at a low level, but using the negative ion detection it is possible to trace and identify such species, which were not detectable even by TLC. Advantage of the low-energy CID fragmentation for low abundance components of the complex ganglioside mixtures in the negative ion mode is to produce clear-cut series of fragment ions for sequencing. Fucosylation analysis carried out for each molecular species by MS/MS permits the clear distinction between sLex and VIM-2 epitope. VIM-2 epitope was expressed in all species with a longer sugar core, while in the short oligosaccharide chain with a sLex only, using biological material at a mid-femtomol level detection.     

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.     

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 CH₃COO^? 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.     

Gangliosides' analysis by MALDI-ion mobility MS

The combination of ion mobility with matrix-assisted laser desorption/ionization allows for the rapid separation and analysis of biomolecules in complex mixtures (such as tissue sections and cellular extracts), as isobaric lipid, peptide, and oligonucleotide molecular ions are pre-separated in the mobility cell before mass analysis. In this study, MALDI-IM MS is used to analyze gangliosides, a class of complex glycosphingolipids that has different degrees of sialylation. Both GD1a and GD1b, structural isomers, were studied to see the effects on gas-phase structure depending upon the localization of the sialic acids. A total ganglioside extract from mouse brain was also analyzed to measure the effectiveness of ion mobility to separate out the different ganglioside species in a complex mixture.     

A glycomics platform for the analysis of permethylated oligosaccharide alditols

This communication reports the development of an LC/MS platform for the analysis of permethylated oligosaccharide alditols that, for the first time, demonstrates routine online oligosaccharide isomer separation of these compounds before introduction into the mass spectrometer. The method leverages a high-resolution liquid chromatography system with the superior fragmentation pattern characteristics of permethylated oligosaccharide alditols that are dissociated under low-energy collision conditions using quadrupole orthogonal time-of-flight (QoTOF) instrumentation and up to pseudo MS(3) mass spectrometry. Glycoforms, including isomers, are readily identified and their structures assigned. The isomer-specific spectra include highly informative cross-ring and elimination fragments, branch position specific signatures, and glycosidic bond fragments, thus facilitating linkage, branch, and sequence assignment. The method is sensitive and can be applied using as little as 40 fmol of derivatized oligosaccharide. Because permethylation renders oligosaccharides nearly chemically equivalent in the mass spectrometer, the method is semiquantitative and, in this regard, is comparable to methods reported using high field NMR and capillary electrophoresis. In this postgenomic age, the importance of glycosylation in biological processes has become clear. The nature of many of the important questions in glycomics is such that sample material is often extremely limited, thus necessitating the development of highly sensitive methods for rigorous structural assignment of the oligosaccharides in complex mixtures. The glycomics platform presented here fulfills these criteria and should lead to more facile glycomics analyses.     

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.     

基于毛细管电泳技术的高灵敏度蛋白质组学技术发展

近年来,蛋白质组学技术在样品前处理、分离技术和质谱检测技术方面获得了快速发展,已经可以实现在几小时内对上万种蛋白的同时定性和定量分析。然而,目前的主流蛋白质组学技术仍无法满足极微量生物样品,尤其是单细胞样品的组学分析需求。毛细管电泳分离技术具有峰宽窄、柱效高、样品用量少等优势,是与高分辨质谱在线联用的理想选择之一。该文评述了集成化和在线样品前处理以及主流的纳升液相色谱-质谱联用系统在高灵敏度蛋白质组学分析领域的发展现状和挑战,认为该领域的重要技术挑战之一在于目前的纳升液相色谱分离已经无法完全匹配现代高分辨质谱超过40 Hz的超高扫描速度,从而导致质谱使用效率的降低。针对上述技术挑战,该文重点探讨了毛细管电泳-质谱联用技术的独特技术优势和潜在发展机遇,主要包括:(1)面向微量酶解多肽样品的高柱效毛细管电泳分离。通过采用毛细管电色谱可以进一步改善毛细管电泳柱容量不足的局限;(2)面向高灵敏度分析的无鞘液/鞘液接口开发;(3)高效毛细管电泳分离与高扫描速度质谱检测的协同化使用。总之,我们预期毛细管电泳-质谱联用技术的进一步发展有望在针对单细胞等超微量生物学样品的蛋白质组学分析中获得更广泛的应...     

Formation of multimeric steroid metal adducts and implications for isomer mixture separation by traveling wave ion mobility spectrometry

Steroid analysis is essential to the fields of medicine and forensics, but such analyses can present some complex analytical challenges. While chromatographic methods require long acquisition times and often provide incomplete separation, ion mobility spectrometry (IMS) as coupled to mass spectrometry (MS) has demonstrated significant promise for the separation of steroids, particularly in concert with metal adduction and multimerization. In this study, traveling wave ion mobility spectrometry (TWIMS) was employed to separate multimer steroid metal adducts of isomers in mixtures. The results show the ability to separate steroid isomers with a decrease in resolution compared with single component standards because of the formation of heteromultimers. Additionally, ion‐neutral collision cross sections (CCS) of the species studied were measured in the mixtures and compared with CCSs obtained in single component standards. Good agreement between these values suggests that the CCS may aid in identification of unknowns. Furthermore, a complex mixture composed of five sets of steroid isomers were analyzed, and distinct features for each steroid component were identified. This study further demonstrated the potential of TWIMS‐MS methods for the rapid and isomer‐specific study of steroids in biological samples for use either in tandem with or without chromatographic separation.     

The potential for clinical applications using a new ionization method combined with ion mobility spectrometry-mass spectrometry

Recently discovered ionization methods for use in mass spectrometry (MS), are widely applicable to biological materials, robust, and easy to automate. Among these, matrix assisted ionization vacuum (MAIV) is astonishing in that ionization of low and high-mass compounds are converted to gas-phase ions with charge states similar to electrospray ionization simply by exposing a matrix:analyte mixture to the vacuum of a mass spectrometer. Using the matrix compound, 3-nitrobenzonitrile, abundant ions are produced at room temperature without the need of high voltage or a laser. Here we discuss chemical analyses advances using MAIV combined with ion mobility spectrometry (IMS) real time separation, high resolution MS, and mass selected and non-mass selected MS/MS providing rapid analyte characterization. Drugs, their metabolites, lipids, peptides, and proteins can be ionized simultaneously from a variety of different biological matrixes such as urine, plasma, whole blood, and tissue. These complex mixtures are best characterized using a separation step, which is obtained nearly instantaneously with IMS, and together with direct ionization and MS or MS/MS provides a fast analysis method that has considerable potential for non-targeted clinical analyses.     

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.     

Copper-coated microsprayer interface for on-line sheathless capillary electrophoresis electrospray mass spectrometry of carbohydrates

A sturdy home-built sheathless CE/ESI-QTOF-MS system was developed and optimized for carbohydrate analysis. The interface and employed methodology provided a simple analytical solution to laborious CE/MS interfacing methods and to problems in characterization of complex carbohydrate mixtures that require high-resolution separation of the components. The CE/ESI interface, feasible in any MS laboratory, consists of a one-piece CE column having the CE terminus in-laboratory shaped as a microsprayer and coated with copper. The CE microsprayer was inserted into an in-house made stainless steel clenching device and the whole assembly was mounted onto a quadrupole TOF mass spectrometer. The analytical potential of the interface in terms of suitability, microsprayer performance, copper coat durability, ionization efficiency, spray stability, and sensitivity was tested first on a simple mixture of standard saccharides, which were separated, resolved, and detected with high separation efficiency. The approach was next assessed for the screening of a biological sample, a complex mixture of O-glycosylated sialylated amino acids from urine of a patient suffering from Schindler disease. Preliminary data allow this method to be considered as one of general applicability in structural glycobiology and glycomics and easy to be implemented for proteomic surveys as well.     

A sialylation study of mouse brain gangliosides by MALDI a-TOF and o-TOF mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) process of sialoglycoconjugates is generally accompanied by different levels of cleavage of sialic acid residues and/or by dehydration, and decarboxylation reactions. Quantitative densitometry of the mouse brain ganglioside (MBG) components separated by high-performance thin layer chromatography (HPTLC) and evidenced by orcinol staining was a basis to verify the ganglioside composition pattern with respect to the relative abundances of individual components in the mixture. A systematic mass spectrometry (MS) sialylation analysis has been carried out to evaluate the feasibility of an axial time-of-flight (a-TOF) MS, equipped with a vacuum MALDI source and an orthogonal-TOF (o-TOF) instrument with an ion source operated at about 1 mbar of N(2). Besides, the esterification by one methyl group of the carboxyl group in sialic acid to increase the stability of the ganglioside species for MALDI MS analysis has been tested and the yield of intact ganglioside species and of the neutral loss of water and carbon dioxide estimated. For the sialylation analysis of native ganglioside mixtures the MALDI o-TOF analysis with 6-azo-2-thiothymine/diammonium citrate (ATT/DAC) as a matrix appears as an optimal approach for ganglioside profiling.     

Congener-specific determination of dioxins and related compounds by gas chromatography coupled to LRMS,HRMS, MS/MS and TOFMS

Their characteristics as persistent organic pollutant and their toxicity (2,3,7,8-TCDD is named as a known human carcinogen) make the dioxins and related compounds a focus of interest in environmental analytical chemistry. In view of the widespread distribution of dioxins in the environment, these compounds must be monitored in several matrices, such as air, effluents, soil, sludge and biological samples. The analytical methodologies are especially difficult owing to the complexity of the mixtures of congeners (210 PCDD/Fs and 209 PCBs) and to the low detection limits required (ppb to ppq). Moreover, time-consuming sample preparation steps are needed owing to the presence of a large number of interfering compounds. The different toxicity of each congener requires the development of congener specific methods. This review of trace dioxin determination by mass spectrometry (MS) includes sample preparation and chromatographic separation. In this Special Feature, the use of different MS techniques such as low-resolution MS (LRMS) and high-resolution MS (HRMS) is discussed in terms of selectivity and sensitivity. The performances of other MS techniques, such as tandem MS (MS/MS) and time-of-flight MS (ToFMS), are compared. Quantification techniques, especially the isotopic dilution method, are also discussed. Conclusions and future perspectives are outlined.     

A 2D reversed-phase × ion-pair reversed-phase HPLC-MALDI TOF/TOF-MS approach for shotgun proteome analysis

The separation of complex peptide mixtures in shotgun proteome analysis using a 2D separation scheme encompassing reversed-phase × ion-pair reversed-phase (IP-RP) liquid chromatography coupled online to electrospray ion trap mass spectrometry (MS) has been shown earlier to be superior in terms of separation efficiency and technical robustness compared to the classically used separation scheme encompassing strong cation exchange × IP-RP-chromatography in shotgun proteome analysis. In the present study, this novel separation scheme was coupled offline to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF-MS for the analysis of the same sample, a tryptic digest of the cytosolic proteome of the bacterium Corynebacterium glutamicum. Compared to the earlier study, the MALDI-based platform led to a significantly increased number of peptides (7,416 vs. 2,709) and proteins (1,208 vs. 468, without single peptide-based identifications), respectively. This represents the majority of all predicted cytosolic proteins in C. glutamicum. The high proteome coverage, as well as the large number of low-abundant proteins identified with this improved analytical platform, pave the way for new biological studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New perspectives for microbial glycolipid fractionation and purification processes

Microbial glycolipids produced from renewable sources are of considerable interest in light of their promising biological activities and surfactant characteristics when compared to petroleum derived surfactants. Intense research efforts are currently being made to reduce their production costs and optimize recovery as selected mixtures through downstream processes. Due to the high complexity of natural glycolipid mixtures, efficient purification techniques are also required to examine the biological mechanisms of individual species towards human systems for their application in health-related areas. This review deals with recent advances in the development of glycolipid extraction, fractionation and purification methods, with a particular focus on solid support-free liquid-liquid separation techniques including centrifugal partition chromatography (CPC) and counter-current chromatography (CCC). These techniques offer promising perspectives for the preparative or large-scale separation of glycolipids from complex crude extracts, mainly because of their flexibility in solvent system selection and applicability to a diversity of structures of any polarity.     

Accessible proteomics space and its implications for peak capacity for zero-, one- and two-dimensional separations coupled with FT-ICR and TOF mass spectrometry

The number and wide dynamic range of components found in biological matrixes present several challenges for global proteomics. In this perspective, we will examine the potential of zero-dimensional (0D), one-dimensional (1D), and two-dimensional (2D) separations coupled with Fourier-transform ion cyclotron resonance (FT-ICR) and time-of-flight (TOF) mass spectrometry (MS) for the analysis of complex mixtures. We describe and further develop previous reports on the space occupied by peptides, to calculate the theoretical peak capacity available to each separations-mass spectrometry method examined. Briefly, the peak capacity attainable by each of the mass analyzers was determined from the mass resolving power (RP) and the m/z space occupied by peptides considered from the mass distribution of tryptic peptides from National Center for Biotechnology Information's (NCBI's) nonredundant database. Our results indicate that reverse-phase-nanoHPLC (RP-nHPLC) separation coupled with FT-ICR MS offers an order of magnitude improvement in peak capacity over RP-nHPLC separation coupled with TOF MS. The addition of an orthogonal separation method, strong cation exchange (SCX), for 2D LC-MS demonstrates an additional 10-fold improvement in peak capacity over 1D LC-MS methods. Peak capacity calculations for 0D LC, two different 1D RP-HPLC methods, and 2D LC (with various numbers of SCX fractions) for both RP-HPLC methods coupled to FT-ICR and TOF MS are examined in detail. Peak capacity production rates, which take into account the total analysis time, are also considered for each of the methods. Furthermore, the significance of the space occupied by peptides is discussed.     

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C⁴D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused‐silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused‐silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.     

Nano-electrospray ionization time-of-flight mass spectrometry of gangliosides from human brain tissue

A general approach for the detection and structural elucidation of brain ganglioside species GM1, GD1 and GT1 by nano-electrospray ionization quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry (MS), using combined data from MS and MS/MS analysis of isolated native ganglioside fractions in negative ion mode and their permethylated counterparts in the positive ion mode is presented. This approach was designed to detect and sequence gangliosides present in preparatively isolated ganglioside fractions from pathological brain samples available in only very limited amounts. In these fractions mixtures of homologue and isobaric structures are present, depending on the ceramide composition and the position of the sialic acid attachment site. The interpretation of data for the entire sequence, derived from A, B, C and Y ions by nanoESI-QTOFMS/MS in the negative ion mode of native fractions, can be compromised by ions arising from double and triple internal cleavages. To distinguish between isobaric carbohydrate structures in gangliosides, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analysed after permethylation in the positive ion nanoESI-QTOFMS/MS mode, providing set of data, which allows a clear distinction for assignment of outer and inner fragment ions according to their m/z values. The fragmentation patterns from native gangliosides obtained by low-energy collision induced dissociation (CID) by nanoESI-QTOF show common behaviour and follow inherent rules. The combined set of data from the negative and positive ion mode low-energy CID can serve for the detection of structural isomers in mixtures, and to trace new, not previously detected, components.