3 Institute for Medical Physics and Biophysics, Biomedical Analysis Department, University of Münster, Robert-Koch-Str. 31, D-48149, Münster, Germany; 4 National Institute for Research and Development in Electrochemistry and Condensed Matter, Timisoara, Romania; and 5 Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Waldeyerstr. 15, D-48149, Münster, Germany
Received on November 15, 2002; revised on May 22, 2003; accepted on May 22, 2003
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Abstract |
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Key words: capillary electrophoresis / CS / DS oligosaccharides / decorin / ESI QTOF CID-VE mass spectrometry / oversulfation
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Introduction |
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The polysaccharide structure of several GAGs is characterized by the appearance of alternating blocks with characteristic epimerization and sulfation patterns, thereby probably forming oligosaccharide sequences with specific binding functions. Among the GAGs, DS, heparin, and heparan sulfate show considerable conformational flexibility due to the presence of L-iduronic acid (IdoA) residues, which change easily between chair and skew conformations (Ferro et al., 1990). Crystal structures of heparin tetra- and hexasaccharides in the presence of FGF-2 indicated that FGF binding stabilizes the 1C4 conformation of the IdoA2S residue involved in binding (Faham et al., 1996
). Another important aspect for the function of GAG chains is the possible existence of several binding domains along a single GAG chain. Such chains could exhibit multivalent properties, facilitating for example receptor dimerization. Considered together, these facts indicate that a detailed knowledge of the fine structure of GAG chains would help interpret the mechanisms required for the manifestation of their biological properties.
Recent reports, mostly dealing with the mass spectrometric analysis of heparan sulfate and CS (Rhomberg et al., 1998; Duteil et al., 1999
; Ruiz-Calero et al., 2001
) and rarely with that of DS (Yang et al., 2000
) have shown that capillary electrophoresis (CE) in combination with electrospray ionization mass spectrometry (ESI-MS) is an accurate and sensitive method that can provide a fair amount of information helpful in the fine structural investigation of GAG oligosaccharides.
We have recently demonstrated the feasibility of an offline CE/ESI-MS and tandem MS (MS/MS) approach in CS/DS oligosaccharide analysis (Zamfir et al., 2002) and shown that by CE/ESI-MS and MS/MS the molecular constitution of CS/DS oligosaccharides obtained from bovine aorta can be determined.
In the present study we describe a further development of our methodology based on CE and ESI-quadrupole time-of-flight (QTOF)-MS/MS toward the structural characterization of CS/DS oligosaccharide mixtures. Decorin from skin fibroblast secretions was chosen as the starting source of the oligosaccharides to be analyzed because decorin is probably the main source of CS/DS saccharides with FGF-2-binding activity in the wound fluid (Penc et al., 1998). Hence, it seems reasonable to expect that the data on the overall composition and the frequency of the appearance of defined domains of the GAG chain of decorin would facilitate further investigations on the biological properties of this abundant proteoglycan. By optimizing the CE and ESI-MS conditions, we succeeded in the structural characterization of up to 12-mer CS/DS oligosaccharides expressing different degrees of sulfation. The combination of enzymatic degradation, CE, and MS allowed the determination of building blocks; determination of the chain size and type of the repeating HexA-HexNAc, HexA-HexNAc(S) units; and the identification of oversulfation patterns. In addition, collision-induced dissociation (CID) conducted at variable energies (VE), developed within the present study, was proven to be a novel potent tool for GAG oligosaccharide sequencing and determination of sulfation patterns. The application of tandem MS under CID-VE conditions to the structural analysis of the pentasulfated hexasaccharide allowed the identification of oversulfated domains with potential relevance for carbohydrateprotein interactions.
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Results |
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Screening of CS/DS oligomers by CE with UV detection
The heterogeneity of the oligosaccharide mixture obtained after chondroitin B lyase digestion and gel permeation chromatography was first demonstrated by CE with UV detection. Ammonium acetate in H2O/MeOH served as a CE electrolyte due to its compatibility with () ESI-MS analysis of carbohydrates (Zamfir and Peter-Katalni, 2001
) and its particular suitability for CE separation and nanoESI-MS detection of CS/DS oligosaccharides (Zamfir et al., 2002
). Under these conditions a series of 11 distinct components, as judged from the UV profile at 214 nm, could reproducibly be eluted from the capillary (Figure 3).
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We have reported previously (Zamfir et al., 2002) that under the CE buffer conditions already mentioned, GAG oligosaccharides of higher molar sulfate content are well separated from nonsulfated species and migrate toward the CE cathode within the shortest time period. For the sake of simplicity and to avoid further sample dilution, we choose to combine the various oligosaccharides in two fractions only. The combination into two fractions was further facilitated by the strict run-to-run reproducibility of the CE/UV detection.
NanoESI-QTOF-MS was used in the negative ion mode to analyze the fractions of CS/DS oligomers separated by CE throughout this study. The CE fractions, analyzed by offline nanoESI-QTOF-MS as will be described, were identified in course of this study as hexa-, octa-, deca-, and dodecasaccharides, bearing one double bond and different numbers of sulfate groups. The mass spectrum of the CE fraction collected within the first 3 min after sample application and corresponding to peaks 17 depicted in the CE/UV pattern is shown in Figures 4 and 5. The assignment of single molecular species detected in this mass spectrum is given in Table I. Obviously, species of higher molar sulfate content were eluted first and detected in this first CE fraction, well separated from the second fraction of nonsulfated species collected after running times from 3 to 10 min.
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Several fully sulfated oligosaccharides were found, for example, the tetrasulfated octasaccharide, represented by an ion at m/z 458.02, assigned to its [M-4H]4 molecular ion and m/z 611.28 assigned to its [M-3H]3 ion, respectively, along with the pentasulfated decasaccharide represented by its [M-4H]4 ion at m/z 572.63 and by the m/z 764.03 representing its [M-3H]3 ion. Also, the trisulfated hexasaccharide ion at m/z 687.38 could be assigned to the [M-2H]2 of the fully sulfated glycoform. The hexasulfated dodecasaccharide was represented by its [M-3H]3 molecular ion at m/z 917.13. Beside the regular CS/DS species containing one sulfate ester group per disaccharide unit, five undersulfated ones were also observed: penta- and tetrasulfated dodecasaccharides, tetra- and trisulfated decasaccharides, and a trisulfated octasaccharide (Figures 4 and 5; Table I).
An important detail of the MS experiment presented in Figure 4 was the detection and identification of three oversulfated CS/DS molecular species: two abundant pentasulfated hexasaccharides assigned to both unsaturated -IdoAGalNAc[GlcAGalNAc]2(5S) and saturated IdoAGalNAc[GlcAGalNAc]2(5S) species, were present as triply charged ions at m/z 511.38 and m/z 517.38, respectively, beside the hexasulfated octasaccharide
-IdoAGalNAc[GlcAGalNAc]3(6S), found as a triply charged ion at m/z 664.89. The triply charged ion at m/z 511.38, assigned to the composition of
-IdoAGalNAc[GlcAGalNAc]2(5S), has been subjected to MS/MS fragmentation analysis by CID at low energy to determine the sequence of this previously not characterized CS/DS oligosaccharide. In this respect, the values of collision energy and collision gas pressure played crucial roles in the control of the fragmentation process, which in this case must allow the cleavage of glycosidic bonds while the cleavage of the sulfate groups has to be avoided.
In previous studies on ESI/CID-MS/MS of CS oligosaccharides (Zaia et al., 2001) CID product ion spectra of regularly sulfated CS di-, tetra-, hexa- and octasaccharides have been acquired. It has been observed that the loss of sulfate during the CID process could be minimized in case of those precursor ions where the charge equaled the number of the sulfate groups. In addition, the internal fragment ion formation of these precursor ions was minimized when the collision energy was set to a predetermined value within the 1020 eV range. According to their protocol, the most efficient fragmentation of the triply charged hexasaccharide (3S) was obtained at collision energy of 17.5 eV. At this value, five fragment ions, namely Y1, Y3, Y5, B3, and B5, were obtained.
This sequencing protocol has been applied by us to the pentasulfated hexasaccharide species detected as a triply charged ion in the first CE fraction. In our case, the Y1, Y2, Y5, and B5 set of product ions was detected, from which the position of the two additional sulfate groups could not have been postulated. Therefore, it appeared necessary to develop another protocol, in which the total ion chromatogram (TIC) was acquired for 15 min under variable values of the collision energy, ranging from 10 to 30 eV. By applying this particular protocol, for which we introduce the term CID-VE, a higher coverage of fragment ions relevant for the oligosaccharide sequencing was obtained. The fragmentation spectrum obtained by CID-VE combining in progress along the entire TIC range is depicted in Figures 69. The cleavage specificity of chondroitin B lyase, whereby a 4,5-double bond in the IdoA moiety at the nonreducing end is formed, was taken in account. Eleven oversulfated, 19 fully sulfated, and 24 undersulfated fragment ions, detected in this experiment, are listed in Table II. From these data it is obvious that under the MS/MS conditions chosen for this experiment, the formation of ions resulting from the cleavage of glycosidic bonds was favored and the loss of SO3- groups was minimized. This can be rationalized by the fact that under the mild MS/MS conditions, the formation of multiply charged fragment ions, which are less prone to sulfate cleavage then those that are singly charged, was enhanced. When the CID conditions were changed during the same experiment to elevated values of collision energy and collision gas pressure, smaller fragment ions, relevant for localization of the sulfate groups along the GAG chain, were generated as well.
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This structural proposal is supported also by the tetrasulfated B4-H2O detected as doubly charged ion at 520.03 and the series of the oversulfated pentasaccharide fragments C5 (4S), C5 (3S), and B5 (3S) detected as doubly charged ions of fairly high abundance at 634.45, 595.04, and 586.09, respectively. Another confirmation is given by the fragmentation pattern from the reducing end, in particular by the GalNAc(1S), observed as Z1 and Y1 singly charged ions at m/z 282.14 and 300.14, and from the nonreducing end, -IdoA(1S) as C1 singly charged ion at 255.34. Beside the monosulfated disaccharide GlcAGalNAc(1S) documented as a singly charged Y2 ion at 476.08, the doubly charged Z3 and Y3 ions at m/z 370.01 and 379.13, respectively, assigned to the disulfated trisaccharide [GalNAcGlcAGalNAc](2S) ion were found. The critical analytical question to be posed concerns, therefore, the position of additional sulfate groups in the trisulfated trisaccharide
-IdoAGalNAcGlcA(3S) ion. The presence of this trisaccharide ion is a strong argument for the structure proposal in which two sulfates are in the GalNAc moiety (GalNAc-4,6-bisulfate) and the terminal IdoA being sulfated as well. Alternatively, instead of doubly sulfated penultimate GalNAc, a partial IdoA(S)GalNAc(S)GlcA(S) sequence could be theoretically possible in which B2 and C2 beside Y4 and Z4 would be diagnostic ions for determination of the sulfate attachment.
In the MS/MS spectrum of the pentasulfated hexasaccharide, the well-known situation of isobaric structures has been encountered for the disaccharide and IdoA-containing sequences discussed. The doubly charged ion at m/z 537.99 assigned to the tetrasulfated tetrasaccharide is overlapping with the singly charged ion at m/z 538.02 (Figure 8) corresponding to the bisulfated disaccharide and the doubly charged ion at m/z 237.56 (Figure 6) assigned to [GlcAGalNAc](1S) is overlapping with the unseparated singly charged ion at 237, which would correspond to the B1 ion of IdoA(1S). According to the combination of the well-resolved MS/MS data of ions carrying more than one charge, the sulfate group localization proposal as depicted in the upper scheme of the hexasaccharide sequence in Figure 10 could therefore be favored.
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Discussion |
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The data obtained from the FGF-2 binding assay revealed that it is the polysaccharide of decorin which binds to FGF-2, whereas for example in case of TGF-ß the core protein represents the binding partner (Hildebrand et al., 1994). Furthermore, we could show that in those chondroitin B lyase-resistant oligosaccharides that are larger than hexasaccharides, the binding sequence may be present. Therefore, it seems likely that certain components of these oligosaccharide structures are of functional importance for FGF-2 signaling. Because these binding structures may represent only a minority of CS/DS building blocks, we tried to elucidate the molecular structures of the full complement of chondroitin B lyase-resistant structures for which an interaction with FGF-2 could be shown. By sequencing single components in such mixtures, the presence of regular or irregular units can be clearly detected.
We have effectively demonstrated here that our three-stage method based on CE, ESI-MS, and -MS/MS is a powerful tool for structural elucidation of GAG chains of decorin prepared from conditioned media of human skin fibroblasts. The success of the method required the development of new conditions for each of the analytical steps involved, the CE separation, the ESI/MS screening, and the sequencing of the GAG species in MS/MS experiments by employing a new approach of CID-VE at variable acceleration energy of the precursor ion. Thus, the CE separation electrolyte ammonium acetate/ammonia, pH 12.0, has been adapted to the requirements for ESI-MS. By CE/UV monitoring, the heterogeneity of the GAG mixture was assessed to detect 11 GAG components, demonstrating a superior separation efficiency under the given conditions. Another interesting information of the MS screening of the CE fractions is that species with high molar sulfate content can clearly be separated from non- or undersulfated ones. We have shown in a previous report (Zamfir et al., 2002) that it may be crucial for a correct determination of the degree of sulfation of a single GAG species to be able to account for artificial loss of sulfate induced by the in-source decay in the MS mode.
In analogy to previous reports about ESI-MS methods for GAG oligosaccharide analysis (Pope Marshall et al., 2001; Zaia et al., 2001
) we have observed that in the negative ESI-MS, the in-source desulfation may be reduced by acquiring spectra under mild values of sampling cone potential, while analyte-buffer clusters should become decomposed effectively. The data shown in Table I indicate that this goal has been met successfully.
Detailed structural characterization was achieved by CID-VE fragmentation of the novel DS-containing hexasaccharide, -4,5-IdoAGalNAc[GlcAGalNAc]2(5S), which rendered a good coverage of fragment ions. According to our MS/MS data, three sulfates are distributed in the IdoAGalNAcGlcA moiety, offering two structural variants: one containing the sulfated IdoA and the disulfation of GalNAc moiety, and the other with the both HexA moieties and the GalNAc each monosulfated. The sequence data confirm the presence of a tetrasulfated tetrasaccharide partial sequence assigned either to the IdoA(S)GalNAc(S)GlcA(S)GalNAc(S) or to the IdoA(S)GalNAc(2S)GlcAGalNAc(S) moiety.
The limits of presently developed strategies can be illustrated also from the work of Kinoshita et al. (2001), who investigated oversulfated hexasaccharides from squid cartilage by fast atom bombardment mass spectrometry and by 1H-nuclear magnetic resonance spectroscopy to detect sulfated GlcA in tetrasulfated and pentasulfated hexasaccharide CS sequences. Using this combined methodology, six possible oligosaccharide sequences could be postulated. Our three-step analysis, combining the CE separation with ESI-MS and a novel approach for CID-VE fragmentation, provides an advantage of a rapid, sensitive, and more precise platform for investigations of fine structure of enzyme-resistant functional domains of CS/DS oligosaccharides, which have not thoroughly investigated yet. The methodology is adaptable to all categories of proteoglycans and is likely to become further improved in terms of sensitivity and identification of isobaric structures by online CE/MS and MS/MS. Elucidation of the oligosaccharide structure(s) responsible for the binding to FGF-2 could eventually be achieved by scaling up the preparation of the different oligosaccharide species obtained by CE and using them for FGF-2 binding assays.
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Materials and methods |
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CE
CE experiments were carried out on a P/ACE 5000 instrument (Beckman, Fullerton, CA) equipped with a UV detector (deuterium lamp, 2 nm wavelength accuracy, 190380 nm wavelength range with filter selection) interfaced with an 486 IBM PS/2 Model 56SX computer running the System Gold dedicated software package to control the instrument and collect experimental data essentially as described before (Zamfir et al., 2002). The CE capillaries were fused silica tubings with 50 µm ID x 375 µm OD and an overall length of 57 cm that were externally coated with polyimide. Each CE capillary was cleaned daily by rinsing with methanol for 30 min and dried under high-pressure air flow for 1520 min. Before sample injection, the capillary was conditioned by flushing for at least 20 min with the running buffer (50 mM ammonium acetate, pH 12.0, in water/MeOH 40:60 [v/v]).
To increase the sample concentration, the aqueous solution of the depolymerized oligosaccharide mixture obtained from the gel filtration chromotography separation was evaporated to a volume of about 80 µl and portioned into two aliquots of 25 µl each and one of 30 µl. One of the 25-µl aliquots was evaporated and the resulting dry substrate dissolved in 15 µl CE buffer for CE/UV experiments. For CE/UV screening, the sample was injected into the CE capillary by applying a constant nitrogen pressure of 0.5 psi for 3 s, equivalent to approximately 12 nl injected volume, and separated at 25 kV forward polarity, which generated a constant current of 33 µA. The UV absorption was monitored at 214 nm for 10 min. The temperature of the capillary cartridge was set at 22°C for all CE experiments.
Offline CE/MS
Offline CE/MS experiments were based on the fraction collection principle as described before (Zamfir et al., 2002). For this experiment, the 30-µl aliquot was dried, redissolved in 7 µl CE buffer, and injected by pressure into the CE capillary for 6 s, resulting in an injected volume of about 20 nl. The separation was performed in the forward polarity at 25 kV. The two CE fractions collected at min 3 and 10 after voltage application were analyzed by negative nanoESI-QTOF-MS and MS/MS.
MS
MS was performed on an orthogonal hybrid quadrupole TOF mass spectrometer (Micromass, Manchester, UK) in the Micromass Z-spray geometry. The QTOF mass spectrometer was interfaced to a PC computer running the MassLynx N.T. software system to control the instrument and to acquire and process MS data.
MS/MS was performed by CID at low energy using argon as a collision gas. Collision energy and gas pressure were readjusted several times during an ongoing MS experiment. The MS/MS spectrum was combined from a TIC acquired within a 1030 eV range of collision energy.
Sample preparation
Decorin (500 µg protein) was prepared from conditioned media of cultured human skin fibroblasts as described previously (Hausser and Kresse, 1999). Its GAG chain was released by a ß-elimination reaction in 200 µl 0.15 M NaOH and 1 M NaBH4 for 20 h at 37°C. The mixture was neutralized with 50% acetic acid, diluted with 1 ml 150 mM NaCl, 20 mM TrisHCl, pH 7.4, and applied to a 0.5 ml DEAE-Tris-Acryl M (BioSepra, Cergy-Saint-Christophe, France) column prepared in a Pasteur pipet. After washing with 1.5 ml 150 mM NaCl, 20 mM TrisHCl, pH 7.4, free GAG chains were eluted with 1.5 ml of 1.0 M NaCl, 20 mM TrisHCl, pH 7.4, dialyzed against water, and lyophilized. Depolymerization of CS/DS (0.5 µmoles of hexuronic acid) was carried out by digestion with 5 mU chondroitin B lyase (Seikagaku Kogyo, Tokyo) in 200 µl 50 mM TrisHCl, pH 8.0, containing 60 mM sodium acetate, 60 mM NaCl, 0.01% bovine serum albumin, and 3 mM NaN3 for 2 h at 37°C. After the first hour of incubation 5 mU of enzyme were added again. Size fractionation of the released oligosaccharides was performed on a Superdex Peptide HR10/30 column (Amersham-Pharmacia, Freiburg, Germany), equilibrated, and eluted in 150-µl fractions with 0.5 M (NH4)HCO3 at a flow rate of 0.5 ml/min and continuous UV detection at 232 nm. The gel filtration column was calibrated with di-, tetra-, and hexasaccharides from testicular hyaluronidase digests of chondroitin sulfate (Kresse et al., 1971
). Fractions eluting earlier than the hexasaccharide standard were pooled and desalted on a prepacked D-Salt column (MW 5000) (Pierce, Rockford, IL). Because rechromatography was avoided to maximize the yield of oligosaccharides, the pooled fraction was expected still to contain contaminating hexasaccharides in addition to higher saccharide species.
FGF-2 binding assay
FGF-2 (Sigma, Taufkirchen, Germany) was brought to a final concentration of 2.5 µg/ml of 100 mM TrisHCl, pH 7.4, containing 50 mM NaCl (coating buffer); 100 µl of this solution were added per well of a MaxiSorp plate (Nunc, Roskilde, Denmark) for about 18 h at 4°C. After washing twice with cold phosphate buffered saline, each well was treated with 200 µl each of 1% bovine serum albumin in phosphate buffered saline for 1 h at 37°C followed by two washings with cold phosphate buffered saline. The wells were incubated for 6 h at 37°C with [35S]sulfate-labeled decorin, decorin-derived intact and partially depolymerized CS/DS chains in 20 mM TrisHCl, pH 7.4, 150 mM NaCl. All these potential ligands of FGF-2 were obtained from metabolically labeled human skin fibroblasts (Hausser and Kresse, 1999) and prepared exactly as described for the nonlabeled proteoglycan species. Unbound [35S]sulfate-label was collected and quantified. After three washings with 100 µl each of phosphate buffered saline, bound [35S]sulfate-labeled material was solubilized with 100 µl 0.5 M NaOH and neutralized with 50 µl 1 M CH3COOH prior to liquid scintillation counting.
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Acknowledgements |
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Footnotes |
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2 To whom the correspondence should be addressed; email: jkp{at}uni-muenster.de
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Abbreviations |
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References |
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