Glycobiology Advance Access originally published online on June 10, 2003
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Glycobiology, 2003, Vol. 13, No. 11 733-742
© 2003 Oxford University Press
Structural investigation of chondroitin/dermatan sulfate oligosaccharides from human skin fibroblast decorin
2,33 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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Hybrid chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan chains, derived from decorin secreted by human skin fibroblasts, were shown to interact with FGF-2, as did oligosaccharides derived therefrom by chondroitin B lyase digestion. In a first attempt to identify the biologically active sequence, a novel protocol for structural analysis of enzyme-resistant oligosaccharides larger than standard trisulfated hexasaccharides was developed. The method bases on capillary electrophoresis (CE) for separating oversulfated species in offline combination with nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the negative ion mode. Under optimized CE and ESI-MS conditions, up to 12-mer oligosaccharides with different degrees of sulfation were identified. A novel tandem MS protocol (CID-VE) was applied to elucidate the structure of a previously undescribed pentasulfated CS/DS hexasaccharide,
-4,5-IdoAGalNAc[GlcAGalNAc]2(5S). In this molecular species, detected as a triply charged ion at m/z 511.38, three sulfates are found in the IdoAGalNAcGlcA moiety offering two structural variants: one containing sulfated IdoA together with a disulfated GalNAc moiety and in the other one both uronic acids, that is, GlcA and IdoA and the amino sugar each carry a sulfate ester group. Key words: capillary electrophoresis / CS / DS oligosaccharides / decorin / ESI QTOF CID-VE mass spectrometry / oversulfation
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Introduction |
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Proteoglycans are a class of highly versatile molecules equipped with recognition markers both on their glycosaminoglycan (GAG) and on their protein moieties. The importance of the recognition structures of the GAG moiety components is best exemplified in case of heparin and heparan sulfate. Heparin and heparan sulfate can interact, for example, with growth factors, chemokines, cytokines, matrix molecules, cell membrane receptors, enzymes, and clotting cascade components (for a review, see Bernfield et al., 1999
; Casu and Lindahl, 2001). Much less is known about potentially active domains of chondroitin/dermatan sulfate (CS/DS). Interactions with heparin cofactor II (Maimone and Tollefsen, 1990; Mascellani et al., 1993) and protein C inhibitor (Priglinger et al., 1994) have been described. DS may interact in place of heparin or heparan sulfate with matrix proteins, like fibronectin (Walker and Gallagher, 1996), cell membrane receptors, and platelet factor 4 (Cella et al., 1992). Furthermore, DS plays a role in the signaling behavior of fibroblast growth factor-2 (FGF-2) (Penc et al., 1998) and hepatocyte growth factor/scatter factor (Lyon et al., 1998) and also regulates WISP-1 function (Desnoyers et al., 2001). Penc et al. (1998) demonstrated that DS from proteoglycans released during wound repair can also activate FGF-2 signaling. However, the nature of the FGF-2 binding domain within DS is not known yet, as is the nature of the core protein from which the interacting DS chain is derived. Another important aspect of CS/DS was reported in studies on tumor metastasis. There are indications that CS/DS may regulate angiogenesis and melanoma cell invasion and proliferation (Denholm et al., 2001). Recently, Kawashima et al. (2002) reported that versican, a large CS/DS proteoglycan, interacts through its CS/DS chains with adhesion molecules L- and P-selectin and that this interplay is sulfation-dependent and requires oversulfated domains of CS/DS.
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 carbohydrate–protein interactions.
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Results |
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Interaction of CS/DS oligosaccharides with FGF-2
[35S]Sulfate-labeled decorin, obtained from the conditioned medium of human skin fibroblasts, is able to interact with FGF-2 in a solid phase binding assay (data not shown). Similar data were obtained when [35S]sulfate-labeled GAG chains obtained by reductive ß-elimination were used in place of the intact proteoglycan as a soluble ligand (Figure 1A). For this assay an average amount of 7–20 nmol GAG oligosaccharides was used as determined by carbazole assay. To discriminate between oligosaccharide domains being enriched either in glucuronic acid (GlcA)- or in IdoA-containing disaccharide units, we digested the GAG chains with chondroitin B lyase and separated the products by gel permeation chromatography (Figure 2).
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According to this protocol, all tetra- and larger oligosaccharides should contain exclusively internal GlcA residues as hexuronic moieties. This was verified by the complete sensitivity of these oligosaccharides toward chondroitin ACII lyase, which, complementary to chondroitin B lyase, specifically attacks GlcA–GalNAc linkages (data not shown). In binding studies the tetrasaccharide and larger oligosaccharides obtained from the chondroitin B lyase digest were still capable to bind to immobilized FGF-2 (Figure 1B), albeit at lower capacity than the intact GAG chains obtained by ß-elimination (Figure 1A). A detailed structural analysis of these oligosaccharides was required to determine specific primary structural features, which might create particular epitopes suited for interaction with FGF-2. The heterogeneous fraction containing oligosaccharides eluted prior to hexasaccharides (fraction under the bar) was used for CE and MS analysis (Figure 2).
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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Structural analysis of the CS/DS oligomers by offline CE/ESI-QTOF-MS and MS/MS
For the structural analysis of GAGs, nanoESI-MS has been shown to be an attractive technique due to the sensitivity and the wealth of structural information that can be obtained by MS/MS (Zaia and Costello, 2001
). However, several technical problems of the ESI-MS screening of GAGs were reported as well. They arise because of the difficulty to ionize long chains, the in-source loss of labile sulfate ester groups, and the complexity of the obtained spectra in which an overlapping of isobaric peaks is frequently encountered. For the ESI-MS screening of complex biological mixtures of nonsulfated and sulfated species, the in-source decay of the sulfate moieties is one of the most severe drawbacks. Although mild ESI source parameters substantially reduce this fragmentation, nevertheless it cannot be suppressed completely. Therefore, in the case of complex GAG mixtures, the assessment of the mixture composition by direct ESI-MS analysis may give rise to incorrect conclusions.
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 1–7 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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The complexity of the sample results from the different chain size of the GAG oligosaccharides present, their CS/DS type according to the chondroitin B lyase cleavage, and their sulfation degree. Molecular ions generated from hexa-, octa-, deca-, and dodecasaccharides were shown to carry between one and three sulfate groups per disaccharide unit. Most of molecular species detected in the spectrum carried one double bond, demonstrating that these oligosaccharide species originated from the nonreducing end generated by the eliminative action of chondroitin B lyase on the GalNAc–IdoA linkages. Therefore, these oligosaccharides can be considered to represent hybrid molecules being derived from CS-rich domains bearing a single DS disaccharide unit at the nonreducing end and being linked to a variable number of CS disaccharide units toward the reducing terminal.
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 10–20 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 6–9. 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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A C3 ion detected as a doubly charged species at m/z 397.08 was diagnostic for the localization of sulfate groups within the hexasaccharide chain. It had been assigned to the trisulfated trisaccharide
-IdoAGalNAcGlcA(3S). The doubly charged B4 ion at m/z 528.96 corresponded to the tetrasulfated tetrasaccharide -IdoAGalNAcGlcAGalNAc(4S) along with the corresponding C4 ion at m/z 537.99, both confirming the position of two additional sulfates in the trisaccharide motif at the nonreducing end (Table II).
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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Determination of molecular characteristics of proteoglycans is an essential prerequisite in understanding their biological functions. The finding that in wound fluid DS, probably released from decorin and/or biglycan, is also an activator for FGF-2 signaling like heparan sulfate (Penc et al., 1998
) indicates the possible importance of discrete domain structures of CS/DS, analogous to the observations that in heparan sulfate certain structural criteria must be met to allow efficient signaling via a ternary complex of heparan sulfate, FGF-2, and the FGF-2 receptor (Padera et al., 1999). However, the nature of the FGF-2 binding domain within DS is not yet known.
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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Methanol, aqueous ammonia solution (32%), and ammonium acetate were obtained from Merck (Darmstadt, Germany) and used without further purification. Distilled and deionized water from Mili-Q water systems (Millipore, Bedford, MA) was used for preparation of the CE buffer and sample solutions. Each solution was filtered through 0.2 µm membranes on disposable filter units purchased from Schleicher & Schuell (Dassel, Germany) and degassed before use. Aqueous sample solutions were dried in a Speed Vac SPD 111V evaporator (Savant, Düsseldorf, Germany). The pH value of the CE buffer was adjusted by a 766 pH-meter Calimatic (Knick, Germany). Fused silica CE capillaries were obtained from BGB Analytik Vertrieb (Essen, Germany). Omega glass capillaries used in nanoESI experiments were purchased from Hilgenberg (Germany) and in-house pulled using a vertical pipette puller model 720 from David Kopf Instruments (Tujunga, CA).
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, 190–380 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 15–20 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 10–30 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 Tris–HCl, 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 Tris–HCl, pH 7.4, free GAG chains were eluted with 1.5 ml of 1.0 M NaCl, 20 mM Tris–HCl, 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 Tris–HCl, 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 Tris–HCl, 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 Tris–HCl, 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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This work has been carried out within the Sonderforschungsbereich (SFB) 492 Extracellular Matrix: Biogenesis, Assembly and Cellular Interaction (Projects Z2, A6, and A9) of the Deutsche Forschungsgemeinschaft. We thank Prof. Dr. Peter Bruckner, Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, for the long-term loan of the CE instrument and Zygmund Budny from the same institute for the help in sample preparation. The ESI-QTOF mass spectrometer was obtained from a HbfG grant (Land Nordrhein Westfalen) to J.P.-K. This article is dedicated to the memory of Professor Hans Kresse, deceased on March 13, 2003.
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Footnotes |
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1 These authors contributed equally to this work
2 To whom the correspondence should be addressed; email: jkp{at}uni-muenster.de
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Abbreviations |
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CE, capillary electrophoresis; CID, collision-induced dissociation; CS, chondroitin sulfate; DS, dermatan sulfate; ESI-MS, electrospray ionization mass spectrometry; FGF, fibroblast growth factor; GAG, glycosaminoglycan; GlcA, glucuronic acid; IdoA, iduronic acid; MS, mass spectrometry; MS/MS, tandem mass spectrometry; QTOF, quadrupole time-of-flight; TIC, total ion chromatogram; VE, variable energy
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