Structural characterization of chondroitin/dermatan sulfate oligosaccharides from bovine aorta by capillary electrophoresis and electrospray ionization quadrupole time-of-flight tandem mass spectrometry

An analytical approach based on high-performance capillary electrophoresis (CE) in conjunction with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) has been developed for providing the basis to obtain new insights into the domain structure of the glycosaminoglycan (GAG) moiety of proteoglycans. The feasibility and performance of the off-line CE/ESI-QTOF-MS approach in GAG oligosaccharide analysis were assessed by screening a chondroitin/dermatan sulfate (DS) oligosaccharide mixture obtained from bovine aorta by enzymatic depolymerization by chondroitin B lyase. The CS/DS mixture was analyzed by CE using 50 mM ammonium acetate, pH 12.0, dissolved in aqueous methanol (2:3; v/v), as a CE carrier. Structural identification of the GAG components was achieved using off-line CE/nanoESI-QTOF-MS and-MS/MS experiments. ESI-QTOF instrumental parameters were found to play an important role in the MS screening of the CE-separated GAG species. By optimizing the ESI conditions, oligosaccharides differing in chain length and degree of sulfation could be detected. The building block composition, the size of the carbohydrate chain, as well as structural features of the repeating HexA-GalNAc, HexA-GalNAc(S) units, have been determined using MS/MS by applying collision-induced dissociation at low energies. Cleavage of GAG chains by chondroitin B lyase occurs with formation of structural markers useful for identification of IdoA-containing domains.     

Combining size-exclusion chromatography and fully automated chip-based nanoelectrospray quadrupole time-of-flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive β-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]? ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.     

Multistage Tandem Mass Spectrometry of Chondroitin Sulfate and Dermatan Sulfate

Chondroitin/dermatan sulfate (CS/DS) is a glycosaminoglycan (GAG) found in abundance in extracellular matrices. In connective tissue, CS/DS proteoglycans play structural roles in maintaining viscoelasticity through the large number of immobilized sulfate groups on CS/DS chains. CS/DS chains also bind protein families including growth factors and growth factor receptors. Through such interactions, CS/DS chains play important roles in neurobiochemical processes, connective tissue homeostasis, coagulation, and cell growth regulation. Expression of DS has been observed to increase in cancerous tissue relative to controls. In earlier studies, MS(2) was used to compare the types of CS/DS isomers present in biological samples. The results demonstrated that product ion abundances reflect the types of CS/DS repeats present and can be used quantitatively. It was not clear, however, to which of the CS/DS repeats the product ions abundances were sensitive. The present work explores the utility of MS(3) for structural characterization of CS/DS oligosaccharides. The data show that MS(3) product ion abundances correlate with the presence of DS-like repeats in specific positions on the oligosaccharide chains.     

Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2‐aminoacridone and subjected to reversed polarity CE‐LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE‐LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE‐LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.     

Capillary electrophoresis-mass spectrometry for glycoscreening in biomedical research

Application of capillary electrophoresis (CE) in combination with mass spectrometry (MS) and tandem MS to glycoscreening in biomedical projects is highlighted. In the first part recent CE-MS experiments by sheath liquid CE and multiple stage MS are reported. Neutral and negatively charged N-glycan mixtures from ribonuclease B and fetuin, high-mannose type N-glycoforms, oligosaccharides from lipopolysaccharides (LPS) of Haemophilus influenzae, polysaccharides of Pseudomonas aeruginosa and Staphylococcus aureus were analyzed. A particular emphasis is devoted to the applicability of novel off- and on-line CE-MS and tandem MS methods for screening of proteoglycan-derived oligosaccharides, glycosaminoglycans (GAGs), such as hyaluronates from Streptococcus agalactiae, chondroitin/dermatan sulfates (CS/DS) from bovine aorta and human skin fibroblast decorin, and heparin/heparan sulfate (HS) from porcine and bovine mucosa. The performance of CE-MS/MS for identification of glycoforms in glycopeptides and glycoproteins is illustrated by experiments performed on complex mixtures from urine of patients suffering from a hereditary N-acetylhexosaminidase deficiency (Schindler's disease) and urine of patients suffering from cancer cachexia. For determination of glycosylation patterns in glycoproteins like enzymes and antibodies by CE/MS, both CE-matrix assisted laser desorption/ionization (MALDI) and CE-electrospray ionization (ESI)-MS were functional. Finally, the potential of CE-ESI-MS strategy in glycolipid analysis is demonstrated for gangliosides from bovine brain for which particular CE buffer conditions are required.     

Glycoscreening by on-line sheathless capillary electrophoresis/electrospray ionization-quadrupole time of flight-tandem mass spectrometry

An analytical approach based on sheathless on-line coupling of capillary electrophoresis (CE) and electrospray ionization (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been developed for providing new insight into the characterization of carbohydrate mixtures. The home-built sheathless CE/     

Gas-phase oligosaccharide nonreducing end (GONE) sequencing and structural analysis by reversed phase hplc/mass spectrometry with polarity switching

Here we describe a technique to obtain all the N-linked oligosaccharide structures from a single reversed-phase (RP) HPLC run using on-line tandem MS in both positive and negative ion modes with polarity switching. Oligosaccharides labeled with 2-aminobenzamide (2AB) were used because they generated good ionization efficiency in both ion polarities. In the positive ion mode, protonated oligosaccharide ions lose sugar residues sequentially from the nonreducing end with each round of MS fragmentation, revealing the oligosaccharide sequence from greatly simplified tandem MS spectra. In the negative ion mode, diagnostic ions, including those from cross-ring cleavages, are readily observed in the MS² spectra of deprotonated oligosaccharide ions, providing detailed structural information, such as branch composition and linkage positions. Both positive and negative ion modes can be programmed into the same LC/MS experiment through polarity switching of the MS instrument. The gas-phase oligosaccharide nonreducing end (GONE) sequencing data, in combination with the diagnostic ions generated in negative ion tandem MS, allow both sequence and structural information to be obtained for all eluting species during a single RP-HPLC chromatographic run. This technique generates oligosaccharide analyses at high speed and sensitivity, and reveals structural features that can be difficult to obtain by traditional methods.     

Analysis of oversulfation in a chondroitin sulfate oligosaccharide fraction from bovine aorta by nanoelectrospray ionization quadrupole time-of-flight and fourier-transform ion cyclotron resonance mass spectrometry

A combination of negative ion nano-electrospray ionization Fourier-transform ion cyclotron resonance and quadrupole time-of-flight mass spectrometry was applied to analysis of oversulfation in glycosaminoglycan oligosaccharides of the chondroitin sulfate type from bovine aorta. Taking advantage of the high-resolution and high mass accuracy provided by the FT-ICR instrument, a direct compositional assignment of all species present in the mixture can be obtained. An oligosaccharide fraction containing mainly hexasaccharides exhibited different levels of sulfation, indicated by the presence of species with regular sulfation pattern as well as oversulfated oligosaccharides with one additional sulfate group. Oversulfation can be directly identified from the high-resolution/high mass accuracy FT-ICR mass spectra according to their specific isotopic fine structure. Location of sulfate groups was analyzed by Q-TOF MS and low-energy CID MS/MS. Tetrasulfated hexasaccharides were analyzed by use of collision-induced dissociation at variable collision energy for an unambiguous assignment of the attachment site of the sulfate groups by minimizing unspecific neutral losses. Cleavage of glycosidic bonds gave rise to B- and C-type ions and their respective complementary Y- and Z-type fragment ions.     

Quantitative determination of the glycosaminoglycan Delta-disaccharide composition of serum, platelets and granulocytes by reversed-phase high-performance liquid chromatography

Seven Delta-disaccharide standards from heparan sulfate/heparin (HS/H) and nine Delta-disaccharide standards from chondroitin/dermatan sulfate (CS/DS) and hyaluronic acid (HA) were derivatized with the fluorophore 2-aminoacridone (AMAC) and separated in two runs each by reversed-phase HPLC with baseline separation and very short run times. This novel method facilitates the separation of the largest number of Delta-disaccharides from both CS/DS/HA and HS/H with one column and buffer system after fluorophore labeling in two runs at present. For the first time nine glycosaminoglycan (GAG) Delta-disaccharides from CS/DS/HA were separated after fluorophore labeling in one run. The limits of quantification (LOQs) were below 0.2 pmol for CS/DS/HA and HS/H Delta-disaccharides. We demonstrated applicability of our method for biological samples. Furthermore, normal ranges of the GAG Delta-disaccharide compositions from platelets and granulocytes were determined for the first time.     

Chip‐based high resolution tandem mass spectrometric determination of fibroblast growth factor—chondroitin sulfate disaccharides noncovalent interaction

Fibroblast growth factor‐2 (FGF‐2) is involved in wound healing and embryonic development. Glycosaminoglycans (GAGs), the major components of the extracellular matrix (ECM), play fundamental roles at this level. FGF‐GAG noncovalent interactions are in the focus of research, due to their influence upon cell proliferation and tissue regeneration. Lately, high resolution mass spectrometry (MS) coupled with chip‐nanoelectrospray (nanoESI) contributed a significant progress in glycosaminoglycomics by discoveries related to novel species and their characterization. We have employed a fully automated chip‐nanoESI coupled to a quadrupole time‐of‐flight (QTOF) MS for assessing FGF‐GAG noncovalent complexes. For the first time, a CS disaccharide was involved in a binding assay with FGF‐2. The experiments were conducted in 10 mM ammonium acetate/formic acid, pH 6.8, by incubating FGF‐2 and CS in buffer. The detected complexes were characterized by top‐down in tandem MS (MS/MS) using collision induced‐dissociation (CID). CID MS/MS provided data showing for the first time that the binding process occurs via the sulfate group located at C4 in GalNAc. This study has demonstrated that chip‐MS may generate reliable data upon the formation of GAG‐protein complexes and their structure. Biologically, the findings are relevant for studies focused on the identification of the active domains in longer GAG chains.     

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS²–MS³) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by β-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Δ-[IdoA-GalNAc]. By optimized CID MS²–MS³, fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Δ-[GlcA-GalNAc]. The site of oversulfation was determined by MS²–MS³, which provided sequence patterns consistent with a rare GlcA-3-sulfate–GalNAc-6-sulfate structural motif.      

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.     

Design of a sheathless capillary eletrophoresis-mass spectrometry probe for operation with a Z-spray ionization source

The construction of a sheathless interface for capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS), for operation with a Z-Spray source on a Micromass Quattro-LC triple quadrupole mass spectrometer is described. Designing the interface involved machining a probe compatible with the setup already in place on the mass spectrometer, i.e., MegaFlow-Z ESI. The probe was made of Lexan with the same dimensions as the ESI probe supplied with the instrument. The electrical connection at the electrospray end of the CE capillary was made possible by gold-coating (sheathless CE-ESI-MS). The probe design as well as the electrical and power supply requirements are described in detail. Experiments were performed using this interface, and CE separations of mixtures containing pmole and sub-pmole amounts of peptides were monitored by on-line MS. For a standard peptide mixture (10(-4) M), separation efficiency was typically characterized by N > 10(4) theoretical plates with S/N > 400. Using the same experimental setup, it was also possible to conduct on-line CE-ESI-tandem MS (MS/MS) experiments on the same peptide mixture, and to determine the sequence of the peptides. MS/MS scan functions for different precursor ions were used either alternately or sequentially and the results from both methods were compared. The possibility of peptide mass mapping was explored, and CE-ESI-MS results were obtained for the digestion products of equine myoglobin. Separation efficiencies and S/N values were similar to those obtained for standard peptides. A complete map of the digestion products was obtained.     

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.     

UPLC-MS/MS detection of disaccharides derived from glycosaminoglycans as biomarkers of mucopolysaccharidoses

Mucopolysaccharidoses (MPSs) are a group of disorders resulting from primary defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Depending on the specific enzyme defect, the catabolism of one or more GAGs is blocked leading to accumulation in tissues and biological fluids. GAG measurements are important for high-risk screening, diagnosis, monitoring treatment efficacy, and patient follow up. The dimethylmethylene blue (DMB) spectrophotometric method commonly used in most biochemical genetics laboratories relies on a non-specific total GAG analysis which has led to false positive results, and even false negative results (mainly for MPS III and IV patients). The main objective of our project was to devise and validate a reliable tandem mass spectrometry multiplex analysis for the urine quantitation of four GAGs (dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)) for an eventual technological transfer to the clinic. The developed methodology is rapid (7 min) and our results showed good intraday and interday precision (RSDs ≤ 8.7%) and accuracy (Biases range: −12.0%–18.4%). Linearity was good (r² > 0.995) for DS, HS, CS, and KS calibration curves. In comparison with the DMB spectrophotometric method, this multiplex tandem mass spectrometry method allows GAG fractionation, thus a differentiation of MPS types, except for MPS I and II which are characterized by the same GAG profile. The devised method is a useful and reliable tool for diagnosis of MPS patients, as well as their monitoring and follow up, as shown by longitudinal studies.     

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: A brand‐new attempt at chondroitin sulfate isomers separation

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of ?10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 μm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.     

Study of the electrical connection mechanism of sheathless interface for capillary electrophoresis‐electrospray ionization‐mass spectrometry

With the combination of high separation ability of capillary electrophoresis (CE) and strong identification ability of mass spectrometry (MS), CE/MS is becoming a powerful tool for polar and ionic analytes analysis. Different interfaces have been developed to enhance the sensitivity and reliability since the first introduction of CE/MS in 1987. A sheathless porous interface based on a new ions transferring electric connection technique was reported to be with high sensitivity and reliability. In this work, a series of optical and electrochemical experiments were designed to study the electric connection process. The results indicated that closing CE electrical circuit and applying MS spray voltage were achieved by the small ions transferring through the interface porous wall. The new electric connection method significantly enhanced the sensitivity, resolution and stability of the CE/MS analysis. The interface was applied in CE/MS detection of morphine and 6‐monoacetylmorphine in urine sample and showed an equal sensitivity to LC/MS. With the significant improvement of sensitivity and stability, the CE/MS with the new interface showed strong potential for the determination of low abundance analytes. Copyright © 2012 John Wiley & Sons, Ltd.     

Influence of charge state and sodium cationization on the electron detachment dissociation and infrared multiphoton dissociation of glycosaminoglycan oligosaccharides

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a useful method for tandem mass spectrometry analysis of sulfated glycosaminoglycans (GAGs). EDD produces abundant glycosidic and cross-ring fragmentations that are useful for localizing sites of sulfation in GAG oligosaccharides. Although EDD fragmentation can be used to characterize GAGs in a single tandem mass spectrometry experiment, SO3 loss accompanies many peaks and complicates the resulting mass spectra. In this work we demonstrate the ability to significantly decrease SO3 loss by selection of the proper ionized state of GAG precursor ions. When the degree of ionization is greater than the number of sulfate groups in an oligosaccharide, a significant reduction in SO3 loss is observed in the EDD mass spectra. These data suggested that SO3 loss is reduced when an electron is detached from carboxylate groups instead of sulfate. Electron detachment occurs preferentially from carboxylate versus sulfate for thermodynamic reasons, provided that carboxylate is in its ionized state. Ionization of the carboxylate group is achieved by selecting the appropriate precursor ion charge state, or by the replacement of protons with sodium cations. Increasing the ionization state by sodium cation addition decreases, but does not eliminate, SO3 loss from infrared multiphoton dissociation of the same GAG precursor ions.     

Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating (HexA-GalNAc sulfate) disaccharides, the functions of which depend on patterns of sulfation and uronic acid epimerization. The correlation of biological activities with structure requires a strategy to determine the sequences of CS oligosaccharides without the need for total isolation. Tandem mass spectrometry has enabled the development of proteomics, based on CID fragmentation of ions produced from complex mixtures of proteolytic peptides, and has the potential for rapid sequencing of CS and other glycosaminoglycan classes. The most challenging aspects of CS sequencing are to distinguish GalNAc residues sulfated at the 4- versus the 6-position and uronic acid epimers. This work describes the utility of (1) reducing terminal derivatives and (2) control of precursor ion charge state for tandem mass spectrometric strategies for determining GalNAc sulfation positional isomers of CS. The capability of tandem MS to differentiate uronic acid epimers is also shown, providing evidence that complete or nearly complete information on CS covalent structure may be obtained using tandem MS.     

Capillary electrophoresis/electrospray ion trap mass spectrometry for the analysis of negatively charged derivatized and underivatized glycans.

The increasing interest in the development of glycoproteins for therapeutic purposes has created a greater demand for methods to characterize the sugar moieties bound to them. Traditionally, released carbohydrates are derivatized using such methods as permethylation or fluorescent tagging prior to analysis by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or direct infusion mass spectrometry. However, little research has been performed using CE with on-line mass spectrometry (MS) detection. The CE separation of neutral oligosaccharides requires the covalent attachment of a charged species for electrophoretic migration. Among charged labels which have shown promise in assisting CE and HPLC separation is the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). This report describes the qualitative profiling of charged ANTS-derivatized and underivatized complex glycans by CE with on-line electrospray ion trap mass spectrometry. Several neutral standard glycans including a maltooligosaccharide ladder were derivatized with ANTS and subjected to CE/UV and CE/MS using low pH buffers consisting of citric and 6-aminocaproic acid salts. The ANTS-derivatized species were detected as negative ions, and multiple stage MS analysis provided valuable structural information. Fragment ions were easily identified, showing promise for the identification of unknowns. N-Linked glycans released from bovine fetuin were used to demonstrate the applicability of ANTS derivatization followed by CE/MS for the analysis of negatively charged glycans. Analyses were performed on both underivatized and ANTS-derivatized species, and sialylated glycans were separated and detected in both forms. The ability of the ion trap mass spectrometer to perform multiple stage analysis was exploited, with MS5 information obtained on selected glycans. This technique presents a complementary method to existing methodologies for the profiling of glycan mixtures.