Structural analysis of permethylated oligosaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry and deutero-reduction

Deutero-reduced permethylated oligosaccharides were analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow ESI source. Under these ionization conditions such derivatives preferentially form sodiated molecular species in addition to protonated molecular species. Under collision-induced dissociation, protonated and sodiated molecular species yield simple and predictable fragment mass spectra. A systematic study was conducted on a series of deutero-reduced permethylated glycans to allow rationalization of the fragmentation processes. MS/MS spectra were characterized by fragments resulting from the cleavage of glycosidic bonds. These fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Furthermore, the substituent 3-linked to a HexNAc unit was readily eliminated. Special attention was devoted to a systematic study of fucosylated glycans. The fucosylated deutero-reduced permethylated glycans were submitted to an acidic hydrolysis, releasing specifically the fucosyl residues. The nascent free hydroxyl groups were subsequently CD3-labelled in order to determine the positions initially bearing the fucosyl residues along the oligosaccharide backbone. This methodology was finally applied to characterize a glycan pool enzymatically released from glycoproteins. The present data show that structural elucidation can be achieved at the 50 fmol level.     

Sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements

Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono- and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components.     

The role of mobile protons in negative ion CID of oligosaccharides

Carbohydrates of all classes consist of glycoform mixtures built on common core units. Determination of compositions and structures of such mixtures relies heavily on tandem mass spectrometric data. Analysis of native glycans is often necessary for samples available in very low quantities and for sulfated glycan classes. Negative tandem mass spectrometry (MS) provides useful product ion profiles for neutral oligosaccharides and is preferred for acidic classes. In previous work from this laboratory, site-specific influences of sialylation on product ion profiles in the negative mode were elucidated. The present results show how the interplay of two other acidic groups, uronic acids and sulfates, determines product ion patterns for chondroitin sulfate oligosaccharides. Unsulfated chondroitin oligosaccharides dissociate to form C-type ions almost exclusively. Chondroitin sulfate oligosaccharides produce abundant B- and Y-type ions from glycosidic bond cleavage with C- and Z-types in low abundances. These observations are explained in terms of competing proton transfer reactions that occur during the collisional heating process. Mechanisms for product ion formation are proposed based on tandem mass spectra and the abundances of product ions as a function of collision energy.     

Microgradient separation technique for purification and fractionation of permethylated N‐glycans before mass spectrometric analyses

Analysis of N‐glycans released enzymatically from patients’ sera or other clinical samples may provide diagnostically and prognostically important information on human disease. Permethylation of these biomolecules simultaneously increases their hydrophobicity and substantially improves their detection parameters in the following mass spectrometric analyses. The overall procedure, from the glycan cleavage to the final mass spectrometric determinations, includes several steps involving extraction, derivatization, and purification. During these steps, certain polymeric contaminants that may have been coincidentally introduced could hamper the final measurements. To understand and counter these interferences and further fractionate or preconcentrate these glycans, we introduce here an effective microgradient chromatographic technique that employs a small reversed‐phase microcolumn connected to a gas‐tight microsyringe delivering a mobile‐phase gradient. After loading the glycan fraction onto the microcolumn, three elution steps are recommended: (1) remove polar contaminants; (2) recover permethylated glycans for either liquid chromatography with electrospray ionization mass spectrometry or matrix‐assisted laser desorption/ionization mass spectrometry; and (3) remove larger polymeric contaminants and regenerate the precolumn. We further demonstrate that the trapped second fraction can be beneficially preconcentrated and further separated to achieve matrix‐assisted laser desorption/ionization mass spectrometric detection of the derivatized N‐glycans up to 6300 Da. The enhanced detection capabilities for tetra‐antennary N‐glycans are of increasing interest in disease biomarker discovery.     

Comparing MALDI-MS, RP-LC-MALDI-MS and RP-LC-ESI-MS glycomic profiles of permethylated N-glycans derived from model glycoproteins and human blood serum

The glycomic profiling of purified glycoproteins and biological specimen is routinely achieved through different analytical methods, but mainly through MS and LC-MS. The enhanced ionization efficiency and improved tandem MS interpretation of permethylated glycans have prompted the popularity of this approach. This study focuses on comparing the glycomic profiling of permethylated N-glycans derived from model glycoproteins and human blood serum using MALDI-MS as well as RP-LC-MALDI-MS and RP-LC-ESI-MS. In the case of model glycoproteins, the glycomic profiles acquired using the three methods were very comparable. However, this was not completely true in the case of glycans derived from blood serum. RP-LC-ESI-MS analysis of reduced and permethylated N-glycans derived from 250 nl of blood serum allowed the confident detection of 73 glycans (the structures of which were confirmed by mass accuracy and tandem MS), while 53 and 43 structures were identified in the case of RP-LC-MALDI-MS and MALDI-MS analyses of the same sample, respectively. RP-LC-ESI-MS analysis facilitates automated and sensitive tandem MS acquisitions. The glycan structures that were detected only in the RP-LC-ESI-MS analysis were glycans existing at low abundances. This is suggesting the higher detection sensitivity of RP-LC-ESI-MS analysis, originating from both reduced competitive ionization and saturation of detectors, facilitated by the chromatographic separation. The latter also permitted the separation of several structural isomers; however, isomeric separations pertaining to linkages were not detected.     

Characterisation and proposed origin of mass spectrometric ions observed 30 Th above the ionised molecules of per-O-methylated carbohydrates

Derivatisation of carbohydrates by permethylation significantly improves the mass spectrometric intensity of carbohydrate-derived ions and allows more readily interpretable fragmentation; in addition, samples are conveniently separated from salts, and larger oligosaccharides are more readily ionised. It has previously been recognised that, in the mass spectra of permethylated carbohydrates, a series of ions indicating species 30 Da larger than the fully methylated carbohydrate molecules are also observed. These species have not been characterised in the literature despite their apparently ubiquitous occurrence in the mass spectra of permethylated carbohydrates. Tandem mass spectrometry (MS/MS) experiments were performed on permethylated carbohydrates and reduced permethylated carbohydrates that exhibit the artefact, demonstrating that the artefact is not reducing terminal specific, and that the artefact can be introduced at any hydroxyl residue. It was further demonstrated through the use of different alkylation reagents that the origin of this artefact group is the alkylating reagent itself. It is proposed that side reactions that occur between the permethylation reagents allow the production of small amounts of iodomethyl methyl ether. This reagent can then compete with methyl iodide for reaction with the carbohydrate -OH groups. The result is partial incorporation of a methoxymethyl moiety instead of a methyl group, detected as '+30' artefact ions.     

On resin synthesis of sulfated oligosaccharides

Sulfated glycans are involved in many biological processes, making well-defined sulfated oligosaccharides highly sought molecular probes. These compounds are a considerable synthetic challenge, with each oligosaccharide target requiring specific synthetic protocols and extensive purifications steps. Here, we describe a general on resin approach that simplifies the synthesis of sulfated glycans. The oligosaccharide backbone, obtained by Automated Glycan Assembly (AGA), is subjected to regioselective sulfation and hydrolysis of protecting groups. The protocol is compatible with several monosaccharides and allows for multi-sulfation of linear and branched glycans. Seven diverse, biologically relevant sulfated glycans were prepared in good to excellent overall yield.

Well-defined sulfated oligosaccharides are important synthetic targets. We present an on resin approach for the synthesis of sulfated glycans with a broad reaction scope that overcomes previous limitations associated with on resin synthesis.     

A computational approach to characterizing bond linkages of glycan isomers using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Glycans commonly exhibit variations in their branching structures and glycosidic bond linkages, in addition to their sequence variation. These glycan features are known to be highly correlated with their biological functions. It is relatively straightforward to deduce the composition and sequence of monosaccharides of a glycan from its tandem mass spectra. However, the characterization of the linkage types of each glycosidic bond is still analytically challenging. In this paper, we present a rank-based discriminative model to differentiate between two types of glycosidic linkages (namely, 1-4 and 1-6) based on the cross-ring fragmentation patterns of the corresponding glycans observed under high-energy collision-induced dissociation (CID). To train our models, we acquired tandem mass spectra for three groups of both native and permethylated linear oligoglucoses using a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI/TOF/TOF) instrument. Based on a 5-fold cross-validation, the prediction accuracies of our model for native glycans are determined to be about 88.4% and 92.9% for 1-6 and 1-4 linkages, respectively. The accuracies determined for permethylated glycans are slightly lower, but comparable: 85.6% and 89.0% for 1-6 and 1-4 linkages, respectively. Our method is implemented as a web-hosted utility, thus making it readily accessible to the public which can be accessed through http://ggdb.informatics.indiana.edu:8080/glycanview.     

Increasing the depth of mass spectrometry-based glycomic coverage by additional dimensions of sulfoglycomics and target analysis of permethylated glycans

Hog or porcine gastric mucin resembles the human source in carrying not only blood group antigens but also the rather rare α4-GlcNAc-capped terminal epitope functionally implicated in protection against Helicobacter pylori infection. Being more readily available and reasonably well characterized, it serves as a good reagent for immunobiological studies, as well as a standard for analytical methodology developments. Current approaches in mass spectrometry (MS)-based glycomic mapping remain vastly inadequate in revealing the full complexity of glycosylation, particularly for cases such as the extremely heterogeneous O-glycosylation of mucosal mucins that can be further sulfated. We demonstrate here a novel concerted workflow that extends the conventional matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) mapping of permethylated glycans in positive ion mode to include a further step of sulfoglycomic analysis in negative ion mode. This was facilitated by introducing a mixed-mode solid-phase extraction step, which allows direct cleanup and simultaneous fractionation of the permethylated glycans into separate non-sulfated and sulfated pools in one single step. By distinct MALDI-MS/MS fragmentation patterns, all previously known structural features of porcine gastric mucin including the terminal epitopes and location of sulfates could be readily defined. We additionally showed that both arms of the core 2 structures could be extended via 6-O-sulfated GlcNAc to yield a series of disulfated O-glycans not previously reported, thus expanding its current glycomic coverage. However, a targeted LC-MSⁿ analysis was required and best suited to dig even deeper into validating the occurrence of very minor structural isomers carrying the Lewis Y epitope implicated by positive antibody binding.     

Enhanced sensitivity of LC-MS analysis of permethylated N-glycans through online purification

Aberrant glycosylation of proteins and lipids has been implicated in many human diseases, thus prompting the need for reliable analytical methods that permit dependable quantification of glycans originating from biological specimens. MS of permethylated glycans is currently employed to monitor disease-related aberrant glycosylation of proteins and lipids. However, enhancing the sensitivity of this type of analysis is still needed. Here, analysis of permethylated glycans at enhanced sensitivity is attained through miniaturized solid-phase permethylation and online solid-phase purification. Solid-phase permethylation method was miniaturized by reducing the amount of sodium hydroxide beads (one-third the original amount) packed in microspin columns. The efficiency of glycan permethylation was not adversely affected by this reduction. Online solid-phase purification of permethylated N-glycans derived from model glycoproteins, such as fetuin, α-1 acid glycoprotein and ribonuclease B, offered more sensitive and reproducible results than offline liquid-liquid and solid-phase extractions. Online solid-phase purification method described here permitted a 75% increase in signal intensities of permethylated glycans relative to offline purification methods. This is mainly due to the minimized sample handling associated with an online cleaning procedure. The efficiency and utility of online solid-phase purification was also demonstrated here for N-glycans derived from human blood serum. Online solid-phase purification permitted the detection of 73 N-glycan structures, while only 63 glycan structures were detected in the case of samples purified through liquid-liquid extraction. The intensities of the 63 structures that were detected in both cases were 75% higher for samples that were purified through the online method.     

Universal proteolysis and MSn for N‐ and O‐ glycan branching analysis

The continually growing list of critical glycosylation‐related processes has made analytical methodology for detailed glycan characterization an area of increasing interest. Glycosylation is a post translational modification of unsurpassed complexity due to the variety of compositions and linkages formed by these biopolymers. Structural characterization of glycan isomers has been achieved using ion trap mass spectrometry and MSⁿ of released, permethylated glycans. However, N‐ and O‐glycans require different sample preparation strategies; and release of the glycans may be hindered, result in degradation of the glycan, and/or produce limited yields of permethylated product. In the current report, we demonstrate universal proteolysis of both N‐ and O‐linked glycoproteins to individual glycoamino acids. These samples were shown to be directly amenable to permethylation and MSⁿ analysis for isomeric structural determination. Universal proteolysis and permethylation provides an identical sample preparation strategy for both classes of glycans that avoids potential pitfalls of commonly used release methods. This methodology should be applicable to all glycoproteins and serve as an alternative to glycan release for MSⁿ branching analysis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Fast atom bombardment mass and tandem mass spectra of protonated and alkali cationized aldobiouronic and pseudoaldobiouronic acid per-O-methyl derivatives

The positive-ion fast atom bombardment mass spectra of permethylated aldo- and pseudoaldobiouronic acids can be used to distinguish these classes of compounds. The collisional-induced dissociation spectra of the [M + H]⁺ ions show fragment ions resulting from glycosidic bond cleavage and successive losses of methanol molecules. These spectra together with those of the [M + H ? MeOH]⁺ oxonium ions allow the identification of the type of interglycosidic linkage. Collisional activation of the relatively stable [M + Na]⁺ ions show many fragmentations which are common to alkali cationized permethylated saccharides. Moreover, fragment ions resulting from two-bond ring cleavage processes yield additional information with respect to the linkage between the hexose and uronic acid units.     

Structural analysis of κ-carrageenan sulfated oligosaccharides by positive mode Nano-ESI-FTICR-MS and MS/MS by SORI-CID

Structural analysis of sulfated oligosaccharides from kappa-carrageenan of up to ten residues (MW >2 kDa) was successfully carried out by positive mode nano-ESI-FTICR-MS together with MS/MS using sustained off-resonance irradiation-collision induced dissociation (SORI-CID). Glycosidic bond cleavage reactions via the B- and Y-types of fragmentation were observed and enabled complete sequencing of the oligosaccharide samples. The positions of the labile sulfate substituents were observable using SORI-CID, enabling the determination of the sequence of the sulfated residues.     

Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans

Heparin-like glycosaminoglycans (HLGAGs) are highly sulfated, linear carbohydrates attached to proteoglycan core proteins and expressed on cell surfaces and in basement membranes. These carbohydrates bind several families of growth factors and growth factor receptors and act as coreceptors for these molecules. Tandem mass spectrometry has the potential to increase our understanding of the biological significance of HLGAG expression by providing a facile means for sequencing these molecules without the need for time-consuming total purification. The challenge for tandem mass spectrometric analysis of HLGAGs is to produce abundant ions derived via glycosidic bond cleavages while minimizing the abundances of ions produced from elimination of the fragile sulfate groups. This work describes the competing fragmentation pathways that result from dissociation of high negative charge state ions generated from HLGAGs. Glycosidic bond cleavage ion formation competes with losses of equivalents of H2SO4, resulting in complex ion patterns. For the most highly sulfated structure examined, an octasulfated tetramer, an unusual loss of charge from the precursor ion was observed, accompanied by low abundance ions originating from subsequent backbone cleavages. These results demonstrate that fragmentation processes competing with glycosidic bond cleavages are more favored for highly sulfated HLGAG ions. In conclusion, reduction of charge-charge repulsions, such as is achieved by pairing the HLGAG ions with metal cations, is necessary in order to minimize the abundances of ions derived via fragmentation processes that compete with glycosidic bond cleavages.     

<Superscript>0,2</Superscript>A<Subscript>n</Subscript> cross-ring cleavage as a general diagnostic tool for glycan assignment in glycoconjugate mixtures

In contrast to proteomics significantly less efficient analytical tools are presently available for high throughput glycomics using mass spectrometry. In this article, a strategy to use the (0,2)A(n) ring cleavage ion at the reducing end of free glycans as a diagnostic ion for assignment of free glycans, in presence of glycopeptides containing similar glycosylation patterns, is presented for rapid distinction in complex mixtures by mass spectrometry. The MS to MS/MS automatic switching, already previously introduced for the on-line LC-MS and CE-MS analysis, is shown in this contribution to be highly functional to obtain diagnostic fragmentation patterns of free glycan precursors in rapid screening of highly complex glycoconjugate mixtures obtained from clinical samples, namely from the urine of patients suffering from congenital disorders of glycosylation. Congenital disorders of glycosylation (CDG) are inherited metabolic diseases based on defects in the glycosylation pathways of glycoconjugates. The urine of CDG patients was reported to contain O-glycans and glycosylated amino acids at concentrations two to three orders of magnitude higher in comparison with the healthy control, characterized by a high degree of heterogeneity concerning the type, number, and values of molecular ions. Using the (0,2)A(n) ring cleavage ion approach by tandem MS, it was possible to sort out free glycans and get them assigned.     

Sodium hydroxide permethylation of heparin disaccharides

Permethylation is a valuable and widely used tool for the mass spectrometry of carbohydrates, improving sensitivity and fragmentation and increasing the amount of information that can be obtained from tandem mass spectrometric experiments. Permethylation of most glycans is easily performed with sodium hydroxide and iodomethane in dimethyl sulfoxide (DMSO). However, permethylation has not been widely used in the mass spectrometry of glycosaminoglycan (GAG) oligosaccharides, partly because it has required the use of the difficult Hakomori method employing the methylsulfinylmethanide ('dimsyl') base, which has to be made in a tedious process. Additionally, the Hakomori method is not as effective as the sodium hydroxide method in making fully methylated derivatives. A further problem in the permethylation of highly sulfated oligosaccharides is their limited solubility in DMSO. This paper describes the use of the triethylammonium counterion to overcome this problem, as well as the application of the sodium hydroxide method to make permethylated heparin disaccharides and their workup to yield fully methylated disaccharides for electrospray ionization mass spectrometry. The ease, speed, and effectiveness of the described methodology should open up permethylation of GAG oligosaccharides to a wider circle of mass spectrometrists and enable them to develop further derivatization schemes in the effort to rapidly elucidate the structure of these important molecules. Permethylation may also provide new ways of separating GAG oligosaccharides in LC/MS, their increased hydrophobicity making them amenable for reversed-phase chromatography without the need for ion pairing reagents.     

ESI-MS differential fragmentation of positional isomers of sulfated oligosaccharides derived from carrageenans and agarans

We have prepared a number of isomeric red seaweed galactan-derivative sulfated oligosaccharides to determine whether there were diagnostic differences among the isomeric mass spectra obtained using ESI CID MS/MS (triple quadrupole instrument). Fragmentation of the single or multicharged molecular ions from di-, tetra-, and hexasaccharides indicated that the relative positioning of the sulfate groups and type of monosaccharide unit affect the rate of cleavage of the glycosidic bonds. We also performed a comparative [M-Na]⁻ fragmentation study of positional isomers of sulfated disaccharides that present all four monosulfation possibilities on the galactopyranosidic ring. In this case, negative-ion ESI CID MS/MS approach gave diagnostic product ions from cross-ring cleavages along with the same main B₁ ion (from sulfated Gal_p), at m/z 241, for all isomers. The isomeric disaccharides were also submitted to increased spray energy conditions inducing in-source fragmentation; preformed B₁ ions were then fragmented to give similar product ions as those found in [M-Na]⁻ analysis. Evaluation of the relative abundances mainly for cross-ring fragment ions at m/z 138, 139, 151, 153 allowed clear distinction among the members of the disaccharide series. The different ratios for m/z 151/153 ions were consistent with the predominance of m/z 153 being related to the cases when the bond involved in the cleavage process links a sulfated carbon. A quadrupole ion trap instrument (MSⁿ analysis) was also utilized to compare the results obtained with the triple quadrupole instrument.     

Liquid chromatography-electrospray mass spectrometry as a tool for the analysis of sulfated oligosaccharides from mucin glycoproteins.

An approach for analyzing sulfated oligosaccharide alditol mixtures by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) is described. Two columns, an amino-bonded column and a porous graphitized carbon column (PGC) were used. Oligosaccharides were eluted with linear gradients of acetonitrile and water, with 5 mM ammonium hydrogencarbonate or formate buffers at a basic pH. The methods were evaluated on a mixture of sulfated oligosaccharide alditols prepared from mucin glycoproteins from pig stomach. Results from LC-ESI-MS of the mixture were compared with the structural information obtained by high energy collision fragmentation using fast atom bombardment tandem mass spectrometry (FAB-MS-MS). The separation ability of the two columns was also tested using a more complex mixture of sulfated oligosaccharides from pig colon, where several isomers were detected. The potential use of in-source collision-induced dissociation (CID) to gain sequence information of sulfated oligosaccharides was also evaluated. The major fragment ions obtained by in-source CID of the trisaccharide Hex-3HexNAcol6-HexNAc6-SO3 were sufficient for assigning the oligosaccharide sequence and the position of the sulfate group within the monosaccharide moiety. The LC-ESI-MS approach should be a valuable tool for characterization of mucin glycosylation and alterations during pathological conditions.     

Identification of N-linked glycosylation sites in human nephrin using mass spectrometry

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.     

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.