Dissociation of peptide ions by fast atom bombardment in a quadrupole ion trap

A new technique for fragmentation of cations and anions of peptides stored in ion traps including radiofrequency devices is described. The technique involves irradiation of peptide ions by a beam of particles generated by a fast atom bombardment (FAB) gun. This irradiation leads to fragmentation of N--C(alpha) backbone bonds (c- and z-fragments) and S--S bonds for cations and C(alpha)-C backbone bonds (a- and x-fragments) for anions of peptides. The fragmentation patterns observed are hypothesized to be due to the interaction of peptide ions with metastable, electronically excited species generated by the FAB gun. Interaction of a metastable atom A* with a peptide n-cation M(n+) leads to the electron transfer from the metastable atom to the polycation through the formation of an ion-pair collision complex A(+.) . . . M((n-1)+.) and subsequent fragmentation of the peptide cation. Thus, for polycations, this metastable-induced dissociation of ions (MIDI) is similar to the phenomenon of electron capture dissociation (ECD). Interaction of A* with an anion leads to the deexcitation of the metastable species and detachment of an electron from the anion. This in turn leads to backbone fragmentation similar to that in electron detachment dissociation (EDD). The MIDI technique is robust and efficient, and it is applicable to peptides in as low charge states as 2+ or 2-.     

Fragmentation of oligoribonucleotides from gas-phase ion-electron reactions

We have recently demonstrated that both electron capture dissociation (ECD) and electron detachment dissociation (EDD) can provide complementary sequence-specific cleavage of DNA compared with collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD). However, EDD is preferred because of more extensive fragmentation at higher sensitivity (due to its negative ion mode operation). Here, we extend the radical ion chemistry of these two gas-phase ion-electron reaction techniques to the characterization of RNA. Compared with DNA, rather limited information is currently available on the gas-phase fragmentation of RNA. We found that the ECD fragmentation patterns of the oligoribonucleotides A6, C6, and CGGGGC are nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. Only limited backbone cleavage was observed in ECD. EDD, on the other hand, provided complete sequence coverage for the RNAs A6, C6, G6, U6, CGGGGC, and GCAUAC. The EDD fragmentation patterns were different from those observed with CAD and IRMPD in that the dominant product ions correspond to d- and w-type ions rather than c- and y-type ions. The minimum differences between oligoribonucleotides suggest that EDD proceeds following direct electron detachment from the phosphate backbone.     

Effect of the N-terminal basic residue on facile Cα-C bond cleavages of aromatic-containing peptide radical cations

Fragmentation of radical cationic peptides [R(G)(n-2)X(G)(7-n)]˙(+) and [R(G)(m-2)XG]˙(+) (X = Phe or Tyr; m = 2-5; n = 2-7) leads selectively to a(n)(+) product ions through in situ C(α)-C peptide backbone cleavage at the aromatic amino acid residues. In contrast, substituting the arginine residue with a less-basic lysine residue, forming [K(G)(n-2)X(G)(7-n)]˙(+) (X = Phe or Tyr; n = 2-7) analogs, generates abundant b-y product ions; no site-selective C(α)-C peptide bond cleavage was observed. Studying the prototypical radical cationic tripeptides [RFG]˙(+) and [KFG]˙(+) using low-energy collision-induced dissociation and density functional theory, we have examined the influence of the basicity of the N-terminal amino acid residue on the competition between the isomerization and dissociation channels, particularly the selective C(α)-C bond cleavage viaβ-hydrogen atom migration. The dissociation barriers for the formation of a(2)(+) ions from [RFG]˙(+) and [KFG]˙(+)via their β-radical isomers are comparable (33.1 and 35.0 kcal mol(-1), respectively); the dissociation barrier for the charge-induced formation of the [b(2)- H]˙(+) radical cation from [RFG]˙(+)via its α-radical isomer (39.8 kcal mol(-1)) was considerably higher than that from [KFG]˙(+) (27.2 kcal mol(-1)). Thus, the basic arginine residue sequesters the mobile proton to promote the charge-remote selective C(α)-C bond cleavage by energetically hindering the competing charge-induced pathways.     

Comparative dissociation of peptide polyanions by electron impact and photo-induced electron detachment

We compare product-ion mass spectra produced by electron detachment dissociation (EDD) and electron photodetachment dissociation (EPD) of multi-deprotonated peptides on a Fourier transform and a linear ion trap mass spectrometer, respectively. Both methods, EDD and EPD, involve the electron emission-induced formation of a radical oxidized species from a multi-deprotonated precursor peptide. Product-ion mass spectra display mainly fragment ions resulting from backbone cleavages of C_α-C bond ruptures yielding a and x ions. Fragment ions originating from N-C_α backbone bond cleavages are also observed, in particular by EPD. Although EDD and EPD methods involve the generation of a charge-reduced radical anion intermediate by electron emission, the product ion abundance distributions are drastically different. Both processes seem to be triggered by the location and the recombination of radicals (both neutral and cation radicals). Therefore, EPD product ions are predominantly formed near tryptophan and histidine residues, whereas in EDD the negative charge solvation sites on the backbone seem to be the most favorable for the nearby bond dissociation.     

Backbone and side-chain cleavages in electron detachment dissociation (EDD)

Ab-initio electronic structure methods are used to explore potential energy profiles pertinent to the fragmentations of gas-phase radicals thought to be formed in the new negative-ion mode EDD mass spectroscopic studies of peptides. Barriers to fragmentation as well as the associated overall energy differences are computed for the observed Calpha-C backbone bond cleavage as well as for side-chain loss for a variety of side chains (valine, arginine, glutamic acid, and tyrosine). It is found that Calpha-C bond cleavage is favored over side-chain loss, although loss of a tyrosine side chain may compete with Calpha-C cleavage because the tyrosine radical formed can delocalize its unpaired electron over its aromatic ring. In addition, it is found that fragmentation of the nitrogen-centered radicals formed in EDD results in cleavage to produce so-called a*/x fragments rather than a/x* fragments both because producing the former involves a significantly smaller barrier and is nearly thermoneutral, while cleavage to yield a/x* is significantly endothermic.     

Observation of an unusually facile fragmentation pathway of gas-phase peptide ions: a study on the gas-phase fragmentation mechanism and energetics of tryptic peptides modified with 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC) and their 18-crown-6 complexes

Various peptide modifications have been explored recently to facilitate the acquisition of sequence information. N-terminal sulfonation is an interesting modification because it allows unambiguous de novo sequencing of peptides, especially in conjunction with MALDI-PSD-TOF analysis; such modified peptide ions undergo fragmentation at energies lower than those required conventionally for unmodified peptide ions. In this study, we systematically investigated the fragmentation mechanisms of N-terminal sulfonated peptide ions prepared using two different N-terminal sulfonation reagents: 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC). Collision-induced dissociation (CID) of the SPC-modified peptide ions produced a set of y-series ions that were more evenly distributed relative to those observed for the SPITC-modified peptides; y(n-1) ion peaks were consistently and significantly larger than the signals of the other y-ions. We experimentally investigated the differences between the dissociation energies of the SPITC- and SPC-modified peptide ions by comparing the MS/MS spectra of the complexes formed between the crown ether 18-crown-6 (CE) and the modified peptides. Upon CID, the complexes formed between 18-crown-6 ether and the protonated amino groups of C-terminal lysine residues underwent either peptide backbone fragmentation or complex dissociation. Although the crown ether complexes of the unmodified ([M + CE + 2H]2+) and SPC-modified ([M* + CE + 2H]2+) peptides underwent predominantly noncovalent complex dissociation upon CID, the low-energy dissociations of the crown ether complexes of the SPITC-modified peptides ([M' + CE + 2H]2+) unexpectedly resulted in peptide backbone fragmentations, along with a degree of complex dissociation. We performed quantum mechanical calculations to address the energetics of fragmentations observed for the modified peptides.     

Characterization of nucleic acid higher order structure by high-resolution tandem mass spectrometry

Mass spectrometry (MS) is extensively used for the identification and sequencing of nucleic acids but has so far seen limited use for characterization of their higher order structures. Here, we have applied a range of different tandem mass spectrometry techniques, including electron detachment dissociation (EDD), infrared multiphoton dissociation (IRMPD), activated ion (AI) EDD, and EDD/IRMPD MS³, in a Fourier transform ion cyclotron resonance mass spectrometer to the characterization of three isomeric 15mer DNAs with different sequences and predicted solution-phase structures. Our goal was to explore whether their structural differences could be directly probed with these techniques. We found that all three 15mers had higher order structures in the gas phase, although preferred structures were predicted for only two of them in solution. Nevertheless, EDD, AI EDD, and EDD/IRMPD MS³ experiments yielded different cleavage patterns with less backbone fragmentation for the more stable solution-phase structure than for the other two 15mers. By contrast, no major differences were observed in IRMPD, although the extent of backbone cleavage was higher with that technique for all three 15mers. Thus, experiments utilizing the radical ion chemistry of EDD can provide complementary structural information compared to traditional slow heating methods, such as IRMPD, for structured nucleic acids.     

Negative-ion electron capture dissociation: radical-driven fragmentation of charge-increased gaseous peptide anions

The generation of gaseous polyanions with a Coulomb barrier has attracted attention as exemplified by previous studies of fullerene dianions. However, this phenomenon has not been reported for biological anions. By contrast, electron attachment to multiply charged peptide and protein cations has seen a surge of interest due to the high utility for tandem mass spectrometry (MS/MS). Electron capture dissociation (ECD) and electron transfer dissociation (ETD) involve radical-driven fragmentation of charge-reduced peptide/protein cations to yield N-C(α) backbone bond cleavage, resulting in predictable c'/z(?)-type product ions without loss of labile post-translational modifications (PTMs). However, acidic peptides, e.g., with biologically important PTMs such as phosphorylation and sulfonation, are difficult to multiply charge in positive ion mode and show improved ionization in negative-ion mode. We found that peptide anions ([M - nH](n-), n ≥ 1) can capture electrons within a rather narrow energy range (~3.5-6.5 eV), resulting in charge-increased radical intermediates that undergo dissociation analogous to that in ECD/ETD. Gas-phase zwitterionic structures appear to play an important role in this novel MS/MS technique, negative-ion electron capture dissociation (niECD).     

Metastable atom‐activated dissociation mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues

Extensive backbone fragmentation resulting in a‐, b‐, c‐, x‐, y‐ and z‐type ions is observed of singly and doubly charged peptide ions through their interaction with a high kinetic energy beam of argon or helium metastable atoms in a modified quadrupole ion trap mass spectrometer. The ability to determine phosphorylation‐sites confirms the observation with previous reports and we report the new ability to distinguish between leucine and isoleucine residues and the ability to cleave two covalent bonds of the proline ring resulting in a‐, b‐, x‐, y‐, z‐ and w‐type ions. The fragmentation spectra indicate that fragmentation occurs through nonergodic radical ion chemistry akin to electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron ionization dissociation mechanisms. However, metastable atom‐activated dissociation mass spectrometry demonstrates three apparent benefits to ECD and ETD: (1) the ability to fragment singly charged precursor ions, (2) the ability to fragment negatively charged ions and (3) the ability to cleave the proline ring that requires the cleavage of two covalent bonds. Helium metastable atoms generated more fragment ions than argon metastable atoms for both substance P and bradykinin regardless of the precursor ion charge state. Reaction times less than 250 ms and efficiencies approaching 5% are compatible with on‐line fragmentation, as would be desirable for bottom‐up proteomics applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Electron detachment dissociation for top-down mass spectrometry of acidic proteins

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72% for Melittin, which lacks carboxylic acid functionalities, to 19% for an acidic 147-residue protein, to 12% for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO(2). Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.     

Elucidation of the fragmentation pathways of azaspiracids, using electrospray ionisation, hydrogen/deuterium exchange, and multiple-stage mass spectrometry

Collision-induced dissociation (CID) mass spectra were generated for azaspiracids using electrospray ionisation (ESI), and hydrogen/deuterium (H/D) exchange was used to ascertain the number and type of replaceable hydrogens in the three predominant azaspiracid toxins. H/D exchange was conveniently achieved using deuterated solvents for liquid chromatography (LC). Using ion-trap mass spectrometry, multiple-stage CID experiments (MS(n)) on the protonated and fully exchanged ions were performed to decipher characteristic fragmentation pathways. The precursor and product ions from azaspiracids lost up to five water molecules from different regions during MS(n) experiments and it was possible to distinguish between the water losses from different molecular regions. These studies confirmed that the first water-loss ion in the spectra of azaspiracids resulted from dehydration at the vicinal diol at C20-C21. Five MS dissociation pathways were identified that resulted from fragmentation of the carbon skeleton of azaspiracids producing nitrogen-containing ions. Two pathways, involving cleavage of the E-ring and C27-C28, gave ions that were found in all azaspiracids. Three pathways, A-ring, C-ring and C19-C20 cleavages, were useful for distinguishing between azaspiracid analogues. The same product ions from backbone fragmentation were also observed using hybrid quadrupole time-of-flight mass spectrometry (QqTOFMS). The fragmentation of the A-ring was the most facile and was exploited in the development of LC/MS(n) methods for the analysis of azaspiracids.     

The effect of fixed charge modifications on electron capture dissociation

Electron capture dissociation (ECD) studies of two modified amyloid beta peptides (20-29 and 25-35) were performed to investigate the role of H* radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H* generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z* ions were still observed in their ECD spectra. Thus, although H* transfer may be important for the NC(alpha) bond cleavage, there also exist other pathways, which would require a radical migration via H* abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides.     

Specific cleavage at peptide backbone Cα-C and CO-N bonds during matrix-assisted laser desorption/ionization in-source decay mass spectrometry with 5-nitrosalicylic acid as the matrix

The use of 5-nitrosalicylic acid (5-NSA) as a matrix for in-source decay (ISD) of peptides during matrix-assisted laser desorption/ionization (MALDI) is described herein. Mechanistically, the decay process is initiated by a hydrogen abstraction from a peptide backbone amide nitrogen by 5-NSA. Hydrogen abstraction results in formation of an oxidized peptide containing a radical amide nitrogen. Subsequently, the C(α)-C bond N-terminal to the peptide bond is cleaved to form an a·/x fragment pair. The C(α)-C bonds C-terminal to Gly residues were less susceptible to cleavage than were those of other residues. C(α)-C bonds N-terminal to Pro and Sar residues were not cleaved by the aforementioned mechanism; instead, after hydrogen abstraction from a Pro or Sar C(α)-H bond, the peptide bond N-terminal to the Pro was cleaved yielding b- and y-series ions. We also show that fragments produced by MALDI 5-NSA-induced ISD were formed independently of the ionization process.     

Electron-induced dissociation of glycosaminoglycan tetrasaccharides

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a powerful tool for examining the structural features of sulfated glycosaminoglycans (GAGs). The characteristics of GAG fragmentation by EDD include abundant cross-ring fragmentation primarily on hexuronic acid residues, cleavage of all glycosidic bonds, and the formation of even- and odd-electron product ions. GAG dissociation by EDD has been proposed to occur through the formation of an excited species that can undergo direct decomposition or ejects an electron and then undergoes dissociation. In this work, we perform electron-induced dissociation (EID) on singly charged GAGs to identify products that form via direct decomposition by eliminating the pathway of electron detachment. EID of GAG tetrasaccharides produces cleavage of all glycosidic bonds and abundant cross-ring fragmentation primarily on hexuronic acid residues, producing fragmentation similar to EDD of the same molecules, but distinctly different from the products of infrared multiphoton dissociation or collisionally activated decomposition. These results suggest that observed abundant fragmentation of hexuronic acid residues occurs as a result of their increased lability when they undergo electronic excitation. EID fragmentation of GAG tetrasaccharides results in both even- and odd-electron products. EID of heparan sulfate tetrasaccharide epimers produces identical fragmentation, in contrast to EDD, in which the epimers can be distinguished by their fragment ions. These data suggest that for EDD, electron detachment plays a significant role in distinguishing glucuronic acid from iduronic acid.     

Side-chain fragmentation of alkylated cysteine residues in electron capture dissociation mass spectrometry

The recent development of novel fragmentation processes based on either electron capture directly or transfer from an anion show great potential for solving problems in proteomics that are intractable by the more widely employed thermal-based fragmentation processes such as collision induced dissociation. The dominant fragmentation occurring upon electron capture dissociation of peptides is cleavage of N-C alpha bonds in the peptide backbone to form c and z* ions. In the case of disulfide-linked peptides, it has also been shown that electron capture on one of the cystine sulfur atoms is favored, resulting in cleavage of the disulfide bond. In this study, we report that electron capture on the sulfur of alkylated cysteine residues is also a dominant process, causing cysteine side-chain loss from z* ions.     

Dissociation pathways of alkali-cationized peptides: Opportunities for C-terminal peptide sequencing

Dissociation pathways of alkali-cationized peptides have been studied using multiple stages of mass spectrometry (MSx) with a quadrupole ion trap mass spectrometer. Over 100 peptide ions ranging from 2 to 10 residues in length and containing each of the 20 common amino acids have been examined. The formation of the [b(n-1) + Na + OH]+ product ion is the predominant dissociation pathway for the majority of the common amino acids. This product corresponds to a sodium-cationized peptide one residue shorter in length than the original peptide. In a few cases, product ions such as [b(n-1) + Na - H]+ and those formed by loss, or partial loss, of a sidechain are observed. Both [b(n-1) + Na + OH]+ and [b(n-1) + Na - H]+ product ions can be selected as parent ions for a subsequent stage of tandem mass spectrometry (MS/MS). It is shown that these dissociation patterns provide opportunities for peptide sequencing by successive dissociation from the C-terminus of alkali-cationized peptides. Up to seven stages of MS/MS have been performed on a series of [b + Na + OH]+ ions to provide sequence information from the C-terminus. This method is analogous to Edman degradation except that the cleavage occurs from the C-terminus instead of the N-terminus, making it more attractive for N-terminal blocked peptides. The isomers leucine and isoleucine cannot be differentiated by this method but the isobars lysine and glutamine can.     

Electron capture dissociation of O-glycosylated peptides: radical site-induced fragmentation of glycosidic bonds

Glycosylation of proteins represents one of the most important post-translational modifications. The structural characterisation of glycoproteins--especially with respect to the determination of the glycosylation site--by direct mass spectrometric methods still remains an elusive goal. We have applied the low energy dissociation method electron capture dissociation (ECD) in a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer to the structural elucidation of mucin-derived peptides glycosylated with glycans of different core types. Capture of an electron by multiply protonated precursor ions [M + nH](n+) resulted in the formation of reduced odd electron radical cations [M + nH](n-1)+*. Subsequent cleavage of the N-Calpha bonds of the peptide chain, mostly without loss of the labile sugar moiety, represents a major fragmentation pathway allowing unambiguous assignment of the glycosylation site. In addition to peptide backbone cleavages, loss of acetyl radicals from the N-acetyl group of the HexNAc glycans is observed. Radical site induced elimination processes of the glycan moieties initiated by hydrogen transfer, from the glycan to the peptide backbone and vice versa give rise to signals in the ECD spectra. The different sugar core types exhibit different fragmentation patterns driven by the stability of the resulting fragments allowing the discrimination of isomeric glycans.     

Cascade Dissociations of Peptide Cation-Radicals. Part 1. Scope and Effects of Amino Acid Residues in Penta-, Nona-, and Decapeptides

Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations.     

Metastable Atom-Activated Dissociation Mass Spectrometry of Phosphorylated and Sulfonated Peptides in Negative Ion Mode

The dissociation behavior of phosphorylated and sulfonated peptide anions was explored using metastable atom-activated dissociation mass spectrometry (MAD-MS) and collision-induced dissociation (CID). A beam of high kinetic energy helium (He) metastable atoms was exposed to isolated phosphorylated and sulfonated peptides in the 3– and 2– charge states. Unlike CID, where phosphate losses are dominant, the major dissociation channels observed using MAD were C_α – C peptide backbone cleavages and neutral losses of CO₂, H₂O, and [CO₂ + H₂O] from the charge reduced (oxidized) product ion, consistent with an electron detachment dissociation (EDD) mechanism such as Penning ionization. Regardless of charge state or modification, MAD provides ample backbone cleavages with little modification loss, which allows for unambiguous PTM site determination. The relative abundance of certain fragment ions in MAD is also demonstrated to be somewhat sensitive to the number and location of deprotonation sites, with backbone cleavage somewhat favored adjacent to deprotonated sites like aspartic acid residues. MAD provides a complementary dissociation technique to CID, ECD, ETD, and EDD for peptide sequencing and modification identification. MAD offers the unique ability to analyze highly acidic peptides that contain few to no basic amino acids in either negative or positive ion mode.     

Electron detachment dissociation of glycosaminoglycan tetrasaccharides

The first application of electron detachment dissociation (EDD) to carbohydrates is presented. The structural characterization of glycosaminoglycan (GAG) oligosaccharides by mass spectrometry is a longstanding problem because of the lability of these acidic, polysulfated carbohydrates. Doubly-charged negative ions of four GAG tetrasaccharides are examined by EDD, collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD). EDD is found to produce information-rich mass spectra with both cross ring and glycosidic cleavage product ions. In contrast, most of the product ions produced by CAD and IRMPD result from glycosidic cleavage. EDD shows great potential as a tool for locating the sites of sulfation and other modifications in glycosaminoglycan oligosaccharides.