Glycobiology Advance Access originally published online on September 15, 2006
Glycobiology 2007 17(1):25-35; doi:10.1093/glycob/cwl046
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Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling
2 Department of Biochemistry, Boston University School of Medicine, MS Resource, 670 Albany Street, Boston, MA 02118, USA
3 Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115
1 To whom correspondence should be addressed; Tel: +1 617-638-6762; Fax: +1 617-638-6760; e-mail: jzaia{at}bu.edu
Received on June 14, 2006; revised on August 11, 2006; accepted on September 5, 2006
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Abstract |
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Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d4 (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.
Key words: cartilage / glycomics / glycosaminoglycan / mass spectrometry
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Articular cartilage is a highly specialized smooth connective tissue, which covers the articulating ends of long bones in a joint (Kuettner 1992
). The function of normal adult joint cartilage resides in its ability to provide resistance to compressive, tensile, and shear forces that occur during normal joint motion. The functioning ability of cartilage is dependent on the maintenance of the extracellular matrix (ECM), which is an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Cartilage integrity depends on a variety of specific and organized interactions between the ECM and chondrocytes. Proteoglycans are major components of the ECM of articular cartilage and provide the tissue with many of its characteristic physicochemical properties, including its ability to produce an osmotic swelling pressure, which enables it to withstand a wide range of compressive loads (). Aggrecan is the major proteoglycan in cartilage matrix by weight and contains approximately 100 chondroitin sulfate (CS) and keratan sulfate (KS) chains per molecule (Hassell et al. 1986). Small leucine-rich proteoglycans (SLRP) that are present in cartilage in equimolar quantities with aggrecan include decorin, biglycan, and fibromodulin.
Sulfated GAGs are linear polysaccharides that consist of repeating disaccharide units that are attached to proteoglycan core proteins on adherent animal cell surfaces and in extracellular matrices (Kraemer 1971; Bernfield et al. 1999; Iozzo 2000; Perrimon and Bernfield 2000). CS is a GAG linked to serine residues in core proteins by way of a xylosyl linker; it consists of repeating disaccharide units of [HexAß/
(1-3)GalNAcß(1-4)], which are polymerized in chains of size varying from 20 to 50 kDa, depending on the core protein, tissue location, age, and disease contexts. The chains consist of mixtures of domains with high or low iduronic acid content with differing patterns of sulfation (Malmström and Fransson 1975). Most types of CS found in higher animals are classified into three main categories: CS type A (CSA), CS type B (CSB, otherwise known as DS, or dermatan sulfate), and CS type C (CSC). CSA is most commonly sulfated (90%) at the 4-position of GalNAc, and CSC is most commonly sulfated (90%) at the 6-position of GalNAc. CSB contains a high percentage of repeats in which the uronic acid residue is epimerized to iduronic acid; a fraction of such repeats are sulfated at the 2-position of iduronic acid. CSB is most often sulfated at the 4-position of GalNAc. In articular cartilage, the most abundant GAGs are CS and KS chains, attached to aggrecan.
Osteoarthritis (OA) is a degenerative joint disease that is characterized by degradation of the cartilage ECM, resulting in fibrillation, irreversible erosion, and eventual failure of the tissue. Maintenance of the ECM requires regulated turnover of aggregating complexes by chondrocytes. Aggrecan and GAGs are actively synthesized and degraded (Collins and McElligott 1960; Ryu et al. 1984; Maroudas et al. 1998), whereas remodeling of the collagen network occurs at a much lower rate (Repo and Mitchel 1971; Maroudas and Venn 1977; Bank et al. 1997). During the development of osteoarthritis, unbalanced proteolytic events give rise to progressive losses of the major CS-bearing regions of aggrecan from the tissue (Mankin et al. 1971; Aigner et al. 1992). Early stages of osteoarthritis involve loss of matrix proteoglycan and type II collagen (Mankin and Lippiello 1970; Mankin et al. 1971; Maroudas and Venn 1977; Scott and Haigh 1985) and an increase in water content in the ECM (Maroudas 1976; Bank et al. 1997). In the later stages of osteoarthritis, there is an inflammatory component to the disease process as ECM breakdown products are released and enter the synovial fluid (Blanco et al. 1998; Abramson et al. 2001; Martin and Buckwalter 2002; Kuhn et al. 2003; Poole 2003; Andriacchi et al. 2004; Sharif et al. 2004).
Previous research has produced evidence that suggests that isomeric CS glycoforms differing in position and degree of sulfation play specific and distinct functional roles during development and disease onset (Mark et al. 1989; Kitagawa et al. 1997; Tufvesson and Malmström 2002). The sulfation patterns of CS chains bound to the cartilage proteoglycan aggrecan have been shown to change with age. Mixtures of 4- and 6-sulfated GalNAc residues were shown to be present in juvenile chicken embryo cartilage, whereas that from human adults contained almost entirely 6-sulfated GalNAc residues (Roughley and White 1980; Belcher et al. 1997; Kitagawa et al. 1997). For cartilage aggrecan, changes in sulfation pattern have been correlated not only with aging and development, but also with the development of osteoarthritis (Shinmei et al. 1992). The 4- to 6-sulfated ratios in synovial fluid are significantly higher for normal joints relative to those from osteoarthritis patients (Sharif et al. 1996). Thus, changes to sulfation ratios occurring with normal aging and with disease development processes are consequences of changes in the posttranslational processing of ECM proteoglycans and to the expression of sulfotransferases by the chondrocytes (Bayliss et al. 1999).
Most of the information on the changes in GAG structure with aging and osteoarthritis derive from disaccharide analysis by chemical or immunological assays. Previous research methods consist of papain digestion of cartilage, followed by ethanol precipitation, exhaustive chondroitinase digestion, and reductive amination with 2-aminopyridine and borane dimethylamine, followed by quantitative analysis by fluorescence-based anion exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis (Plaas et al. 1997, 1998). Previous methods have also included GAG concentration by ultrafiltration (Hickery and Bayliss 1998) and ultrafiltration followed by an additional step in which GAGs are released from the core protein by ß-elimination (Lauder et al. 2000; Huckerby and Lauder 2001). In addition, there is no information on the expression of GAG epitopes larger than trisaccharides in cartilage. Mass spectral (MS) analysis carries the advantage that the compositions and sulfation patterns of extended oligosaccharides can be determined (Zamfir et al. 2002, 2003). An LC-tandem MS method has been applied to the analysis of CS/DS glycoforms of purified GAG and proteoglycan samples (Hitchcock et al. 2006). This method enables determination of sulfation and epimerization of CS/DS tetrasaccharides in a single measurement.
The goal of the present work is to develop methods for MS-based glycomics of cartilage tissue. An MS-compatible method for extraction of GAGs from unfixed cartilage is described. This method is then used with stable isotope labeling to profile the distribution of GAGs in bovine and human cartilage explants.
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An MS-compatible GAG extraction method
An optimized sample workup procedure for the extraction of GAGs from intact cartilage for MS analysis has been developed, as outlined in Figure 1. Explants of juvenile bovine shoulder and adult human knee cartilage were digested with papain and the GAG content measured using a dimethylmethylene blue (DMMB) assay (Table I). Methods published for chromatographic analysis of GAG disaccharides entail digestion of cartilage with papain, ethanol precipitation, and chondroitinase depolymerization (Plaas et al. 1997
, 1998). Data on juvenile bovine shoulder joint disaccharides purified in this manner (Figure 2A) show high levels of noise in the electrospray ionization (ESI) mass spectrum and the absence of peaks corresponding to the target molecules. Owing to this high level of noise, it was necessary to develop a method for the workup of GAGs from intact cartilage tissue that is compatible with MS. For samples purified by the described procedure, a C18 solid-phase extraction step was added to remove hydrophobic protein fragments and lipids. The hydrophilic GAGs were recovered after washing with 0.1% acetic acid. The sample was then applied to a strong anion exchange column in the presence of 50 mM sodium phosphate, pH 3.5, under which conditions GAGs remain negatively charged and able to bind. The GAGs were eluted with a 1 M NaCl solution and precipitated with four volumes of ethanol. The sample was then exhaustively digested with chondroitinases ABC, ACI, and B and subjected to MS analysis. The additional steps produced an approximate 10-fold decrease in MS noise and a strong signal representing a GAG disaccharide (Figure 2B). The [M-H]– GAG disaccharide ion is represented by the peak at m/z 458. Thus, the use of both reversed-phase and strong anion exchange solid-phase extraction steps enables detection of GAG depolymerization products from intact cartilage for MS analysis.
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Quantification of juvenile bovine cartilage
Following optimization of the extraction protocol, GAGs extracted from cartilage were analyzed by ESI-LC/MS/MS. Cartilage samples were purified using steps 1–7 (Figure 1) and then divided into aliquots of 10 µg GAG-equivalent on the basis of the DMMB assay of the papain digest (Burkhardt et al. 2001
). Three such aliquots were analyzed for each cartilage papain digest. A 10% fraction was digested exhaustively using chondroitinases ABC, AC1, and B and derivatized using 2-aminoacridone (AMAC) for capillary electrophoresis-laser-induced fluorescence (CE-LIF) disaccharide analysis (Militsopoulou et al. 2002). The remaining 90% of the sample was digested to 30% completion using all three chondroitinases, as determined by absorbance at 232 nm. The resultant mixture, consisting of dimer, tetramer, and hexamer oligosaccharides, was reductively aminated with 2-anthranilic acid-d4 (d4-2AA) (Bigge et al. 1995). Prior to MS analysis, each sample of d4-2AA derivatized oligosaccharides was mixed with 3 µg of the d0-2AA derivatized CSA oligosaccharides, termed the reference standard. The ESI mass spectrum of a mixture of light (d0) CSA oligosaccharides and heavy (d4) derivatized tetrasaccharides of juvenile bovine cartilage donor A explant 1 is shown in Figure 2C. The d4-2AA-[M-2H]2– GAG tetrasaccharide ion is represented by the peak at m/z 520.4. This tetrasaccharide consisted of a mixture of CSA-like (GlcAß3GalNAc4Sß4), CSB-like (IdoA3GalNac4Sß4), and CSC-like (GlcAß3GalNAc6Sß4) glycoforms.
Tandem mass spectra were acquired on the 2-AA labeled tetrasaccharides extracted from juvenile bovine cartilage donor A explant 1. The mixture composition was determined from the abundances of three diagnostic product ions (Y11–, Y32–, and [M-H-SO3]2–, respectively) (Hitchcock et al. 2006) (Figure 3A). The percent total ion abundances of the heavy form of the three diagnostic ions, labeled in the figure, were calculated and inserted into a system of three equations and three unknowns (Desaire and Leary 2000a, 2000b; Leary and Saad 2003) using coefficients calculated from the ion abundances for purified CSA, CSB, and CSC standards. The percentages of the isomeric glycoforms of CSA, CSB, and CSC extracted from intact juvenile bovine cartilage donor A explant 1 were calculated as the mean values for the three aliquots and were found to be 57.9%±0.9, 2.0%±0.5, and 40.1%±1.1, respectively. The total experimental error for measurement of each glycoform was low and within the range previously described for this method. Extracted CS GAGs from three additional intact bovine cartilage tissues from one individual, labeled juvenile bovine B-1, B-2, and B-3, were analyzed and the results are listed in Table II. The percent compositions for CS glycoforms differed significantly between juvenile bovine individual A (57.9% CSA-like) and B (39.4–43.2% CSA-like). These changes are consistent with the conclusion that the samples were collected from individuals of different juvenile ages.
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Quantification of adult human cartilage
Papain digests of adult human knee cartilage were divided into aliquots of 10 µg GAG equivalent using DMMB assay results. Three aliquots were extracted (Figure 1) and analyzed by CE-LIF and ESI-LC/MS/MS with an internal standard, in the same manner as that of the juvenile bovine cartilage. A representative LC-tandem mass spectrum of adult human cartilage donor A explant 1 is shown in Figure 3B. A series of adult human cartilage explants (13 explants from 5 donors) was used to demonstrate the usefulness of this analytical method for human samples. The percentages of the isomeric glycoforms of CSA, CSB, and CSC determined for each intact adult human cartilage sample are listed in Table II.
The mean percentages of the isomeric glycoforms of CSA, CSB, and CSC in the group of adult human cartilage samples were 8.5%±3.1, 0.9%±0.3, and 90.4%±3.2 (Table II). The average standard deviation among three aliquots of each sample did not vary with the initial weight of the tissue explant and was essentially the same (1.7% of total glycoforms) for CSA-like and CSC-like. For the abundant CSC-like glycoforms, the high degree of reproducibility resulted in a very low coefficient of variation (mean %CV=1.9) (Figure 4). For the lower abundance CSA-like glycoforms, the coefficient of variation was greater (mean %CV=21.5). The greater variance for CSA was likely due to the low abundance of that glycoform in adult human cartilage, and not inherent in measurements of CSA, because the variance was very low for all compositions of >20% abundance, irrespective of glycoform. Nonetheless, it was still possible to discern differences among samples that were due to biological variation, rather than experimental variation.
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Glycoform abundances were compared among multiple explants from the same donor. Very similar levels of CSC were measured in all explants from three of the donors and in three of four explants from another donor (Figure 5). Abundances of CSA-like glycoforms in explants 2, 3, and 4 from human donor A (Table II) were very close in magnitude (average of 6.2%). Using the calculated %CV for CSA, the minimal discernable difference between 6.2% CSA-like and another sample is ±1.3% (i.e. 21.5% of 6.2). Therefore, the range of values within experimental variation is 4.9–7.5% CSA-like. The measured abundance for explant 1 (16.6% CSA-like) exceeded that range. It is concluded that the differences between human donor A explant 1 and 2–4 were not due to experimental variation.
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The average DS (CSB-like) content in juvenile bovine cartilage donors is 1.9%±1.0, whereas that in human articular cartilage donors is 0.9%±0.4 (Table II). Owing to their low abundances, the mean %CV for DS was relatively high (50.3%) and it cannot be concluded that the differences in CSB-like expression levels between the tissue types reflect biological variation. Although aggrecan is the major proteoglycan by weight in cartilage, other proteoglycans are present in equimolar amounts (Poole et al. 1996
). Decorin and biglycan are members of the family of small leucine-rich repeat proteoglycans (Neame et al. 1989) which bind to collagen and have been shown to bind other connective tissue macromolecules (Lewandowska et al. 1987). It has previously been reported that native GAGs from articular cartilage decorin have 31.1%±0.2 CSB-like glycoform content (Miller et al. 2006). Thus, it appears that decorin contains a different pattern of epimerization than do the bulk of CS/DS chains in articular cartilage, deriving from aggrecan.
Disaccharide analysis by CE-LIF
It is useful to compare the LC/MS/MS glycoform quantification results with established methods for disaccharide analysis of the same samples. Previous reports of disaccharide analysis techniques described a procedure in which samples were digested with papain, precipitated with ethanol, chondroitinase digested, and analyzed by fluorescence-based anion exchange HPLC or capillary electrophoresis (Plaas et al. 1997; Hickery and Bayliss 1998; Plaas et al. 1998; Lauder et al. 2000; Huckerby and Lauder 2001). In the present work, the same GAG extraction procedure has been used for disaccharide analysis as with the MS samples. Disaccharide analysis of a 10% fraction of each cartilage aliquot was achieved by exhaustive depolymerization of the whole polymer chain using combined chondroitinase ABC, chondroitinase AC1, and chondroitinase B digestion, followed by fluorescent derivatization using AMAC and CE-LIF analysis of the disaccharide compositions. Migration times of 
-disaccharides generated from cartilage-extracted CS GAGs were verified against those of AMAC-derivatized commercially purified disaccharide standards. An AMAC-derivatized tri-sulfated disaccharide derived from heparin (HSIS) was used as the internal standard for the CS runs because it has a unique migration time compared with the CS disaccharide. The -disaccharide compositions were calculated using the CE fluorescence peak areas, normalized to the area of the internal standard. Table III lists the -disaccharide compositions of the whole polymer chain of every cartilage sample analyzed within this study.
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Juvenile bovine cartilage donor A is shown to contain 57.5%±2.0
HexAß3GalNAc4S (di4S), 39.1%±2.4 HexAß3GalNAc6S (di6S), and 3.4%±0.8 HexAß3GalNAc (di0S), whereas human cartilage sample A explant 1 contains 14.9%±0.8 di4S and 85.1%±0.8 di6S (Table III). Formation of -disaccharides destroys information on the uronic acid epimerization; thus, the distinction between iduronic acid (IdoAGalNAc4S) and glucuronic acid (GlcAGalNAc4S) repeats is lost.
The %CV for the CE-LIF disaccharide analysis is similar to those for the MS measurements (Figure 4). The mean %CV of all disaccharide measurements by CE-LIF was 13.6% for di4S and 2.1% for di6S. Very similar levels of di6S were measured in all explants from each juvenile bovine donor and all but one explant from adult human donors. Abundances of di4S glycoforms in human explants 2, 3, and 4 from human donor A (Table III) were very close in magnitude (average of 9.2%). Using the calculated %CV for di4S, the minimal discernable difference between 9.2% di4S and another sample is ±1.3% (i.e. 13.6% of 9.2). Therefore, the range of values within experimental variation is 7.9–10.5% di4S. The measured abundance for human donor A explant 1 (14.9% HexA-GalNAc4S) exceeded that range. We conclude that the differences between explant 1 and 2–4 were not due to experimental variation.
The mean %CV values from CE-LIF data were comparable with the mean %CV values of glycoform measurements by LC-MS/MS. Direct comparison of LC-MS/MS with CE-LIF data (Figure 6) showed very significant correlations (P<1x10–12) for both CSA/di4S and CSC/di6S. The mean %CV for MS was comparable with the mean %CV for CE-LIF; therefore, the two methods provide equivalent results, even at low glycoform abundance.
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Discussion |
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The biology of the cartilage matrix presents technical challenges for the analysis of matrix GAGs. Low cellularity, zonal and regional variations within the joint, especially in disease, impede quantification of subtle or focal biochemical alterations. Improved analytical methods are therefore of great benefit to identify changes in GAGs that occur with injury, age, or osteoarthritis. MS is a valuable analytical tool for structural and quantitative analyses of biomolecules, such as GAGs, because it offers high analytical versatility, sensitivity, and precision. Thus, it seemed advantageous to create an MS-based extraction method for the analysis of cartilage GAG glycoforms.
For studies of chondrocyte physiology, we use cartilage that is discarded from subjects undergoing total joint replacement for osteoarthritis. Although the cartilage is not normal, the nonweight-bearing areas often have less damage and the joint is not overtly inflamed (compared with rheumatoid arthritis or traumatic fracture). The number and type of assays that can be run on cartilage and chondrocytes obtained from a single donor are limited because the amount of retrievable cartilage is small (<10 g) (Eid et al. 2006; Shortkroff and Yates 2006). In this study, results were obtained from explants weighing 
13 mg.
With the methods reported here, structural information on GAGs can be obtained from focal lesions and surrounding areas. For most of the human cartilage used in this study, explants weighing 10–100 mg were shaved from similarly graded areas of each donor's tibial plateau and femoral condyles. The precise location from which each explant was taken from is not known, because shavings from each donor were pooled before single explants were separated for glycoform analysis. For one donor, the glycoform abundances were similar in three explants taken from a grade 1 region and different in one explant that was taken from a grade 3 region. It is tempting to conclude that glycoform differences related to the extent of degeneration. Grade 3 explants from another donor, however, showed glycoform abundances that were similar to the less-damaged cartilage from other donors. We cannot determine the intersubject variability inherent with age, gender, and tissue degeneration with this sample series (n=5 donors).
Negative mode ESI in the presence of ammonium salts has proved to be effective for the analysis of sulfated carbohydrates (Chai et al. 1998; Kim et al. 1998; Zaia and Costello 2001). Intact negative ions are formed without in-source fragmentation, thereby allowing tandem mass spectra to be acquired through the use of collision-induced dissociation (CID) (Zamfir et al. 2003). Tandem MS produces structural and quantitative information about sulfated GAGs, by virtue of the abundances of specific diagnostic product ions. Thus, it is possible to distinguish among the three isomeric forms of CS/DS on the basis of 2-AA-tetramer product ion abundances (Hitchcock et al. 2006). Following the extraction of GAGs from the intact cartilage tissue, this analytical tool can now be applied to studies of cartilage development and disease processes in which there is evidence that sulfation and epimerization levels of matrix GAGs change.
The method for extraction of GAGs from unfixed intact cartilage tissue samples differs significantly from that used previously for analysis of purified proteoglycan samples (Hitchcock et al. 2006). The tissue is disrupted using papain digestion and following ethanol precipitation; it appears that cellular debris, for example DNA, RNA, and protein fragments, is not completely removed from the cartilage sample and results in a high level of MS noise. The addition of a reversed-phase–solid-phase extraction step aids to remove protein fragments and other biomolecules. The combination of reversed-phase and strong anion exchange solid-phase extraction steps to the workup procedure enabled analysis of GAGs from unfixed tissue using MS. The LC/MS methodology used herein provides the advantage that oligosaccharide sulfation and epimerization patterns are measured simultaneously using an online platform (Hitchcock et al. 2006).
Several age-related increases in the ratio of 6- to 4-sulfated GalNAc residues in articular cartilage CS have been reported (Roughley and White 1980; Belcher et al. 1997; Kitagawa et al. 1997; Bayliss et al. 1999; Lauder et al. 2001). In juvenile articular chick embryo cartilage, mixtures of 4- and 6-sulfated GalNAc residues are known to be present in similar amounts (Roughley and White 1980; Kitagawa et al. 1997). Previous research has shown that 6S/4S ratios vary considerably with age, ranging from 1.0 in fetal to 3.9 in 12-year-old humans (Roughley and White 1980). The nonsulfated disaccharide was shown to be present as a minor component ranging from 5% to 10% of the composition.
Although age-related changes in the proportion of 4- and 6-sulfated GAGs have been described in human articular cartilage, these have been carried out on a relatively small number of specimens from a mixture of joints. The cartilage tissues investigated in this research originated from adult human knee joints and juvenile bovine shoulder joints. The MS results presented herein on GAGs from different explant locations of cartilage in different species agree with the previous findings for the age-related compositions of GAGs in cartilage tissues. Although species-related differences in composition may exist, the results of these studies are consistent with a decrease in 4-sulfation during development and aging. The sulfation profile of CS, both at different positions along a chain and at different ages, is of considerable interest, because some authors have proposed that determination of average 4- or 6-sulfation levels may be an appropriate marker of articular cartilage turnover and of osteoarthritis (Shinmei et al. 1992).
This method is highly sensitive, with excellent intra- and intersample reproducibility. Even for low-abundance glycoforms, this approach is able to discern between samples that differ by as little as 3% of the total composition. The CS disaccharide compositions of adult human knee cartilage reported here are in excellent agreement with an extensive analysis performed by Bayliss et al. (1999). In that study, articular cartilage was harvested from the entire surface of femoral condyles from subjects undergoing surgical treatment for bone tumors, and disaccharide composition analysis was performed to measure the overall abundance of di4S and di6S. Cartilage obtained from subjects over the age of 40 showed di6S that ranged from 70–90%. In the same study, disaccharide compositions were mapped throughout the articular surface of the femoral condyle and tibial plateau. Although only one adult subject was characterized, the distribution of di6S was surprisingly consistent throughout the joint, with 10 of 12 locations showing a pattern of approximately 70% at articular surfaces, which increased to 80–85% within 500 µm below the surface (Bayliss et al. 1999). Only the posterior tip of the lateral condyle and the distal medial condyle on the femur showed a greater abundance of di4S within the first 500 µm from the articular surface. In addition, comparisons of osteoarthritic cartilage with normal cartilage in human (Plaas et al. 1998) or horse knees (Brown et al. 1998) show that internal disaccharide 4- and 6-sulfation does not change, although there is a decrease in sulfation of terminal disaccharides. The disaccharide compositions for all of the human cartilage samples reported here are within the ranges defined in those previously published studies. Moreover, the replicate samples taken from individual donors showed high reproducibility.
This work demonstrates an MS-compatible method for the extraction of GAG oligosaccharides from unfixed cartilage tissue. The method is sensitive and reproducible enough to determine biological variation in different topological positions and zones. Results were shown for the abundant CS/DS-disulfated tetramers. The same methodological principles are applicable to analysis of lower abundance compositions, including over- and under-sulfated sequences and saturated oligosaccharides containing the nonreducing chain termini. In addition, the principles are applicable to the analysis of longer oligosaccharides. Efforts are underway to reduce the scale of the chromatography to allow for analysis of these lower abundance compositions.
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Materials and methods |
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CSA (GlcA, GalNAc-4-sulfate), CSB (IdoA, GalNAc-4-sulfate), CSC (GlcA, GalNAc-6-sulfate), and chondroitinase ABC, B, and AC1 were obtained from Seikagaku America/Associates of Cape Cod (Falmouth, MA). Purified
-disaccharide standards were purchased from V-labs (Covington, LA). The reagents AMAC and d0-2AA were purchased from Fluka Chemika (Buchs, Switzerland), sodium cyanoborohydride and sodium borohydride were from Aldrich Chemicals Co. (St Louis, MO), and 2-anthranilic-3,4,5,6-d4 acid (d4-2AA) was from C/D/N Isotopes (Quebec, Canada). Cellulose packing material Micro Spin Columns and strong anion exchange packing material Micro Spin Columns were purchased from Harvard Apparatus (Holliston, MA). Pepclean C18 reversed-phase spin columns were purchased from Pierce (Rockford, IL).
Articular cartilage
Bovine cartilage was removed from shoulder joints of young calves under aseptic conditions (Yates et al. 2005). The shoulder joint was exposed and full thickness cartilage fragments were dissected from the proximal end of the humerus with a no. 10 scalpel blade. An adjacent fragment was dissected for histology.
Human cartilage was obtained under an institutional review board-approved protocol for discarded tissue (total knee arthroscopy for osteoarthritis). The tibial plateau and femoral condyles were retrieved from the operating room within 3 h after excision from the joint. Human cartilage samples used in this study were collected from explants that had been prepared for chondrocyte isolation (Shortkroff and Yates 2006) as follows: the tibial plateau was evaluated grossly for the extent of wear using a four-point grading system, where 0 describes a normal, smooth glistening surface with appropriate cartilage thickness for the site, 1 has slight surface changes with minimal thinning, 2 has moderate fibrillated and dimpled surface with moderate thinning (50–80% of normal), and 3 shows distinct loss of cartilage with highly fibrillated surface and severe thinning (<25% of normal). Full-thickness cartilage fragments (explants) were shaved from the most normal-appearing (lowest grade) regions of the tibial plateau and from regions of the femoral condyle with the equivalent grade. The explants were weighed and either processed immediately or frozen. For this study, 13 explants (median weight, 28 mg), ranging in grade from 1–3, were collected from five donors (three men and two women, aged 62–81) (Table I). For human donor A, it was possible to collect explants from regions of the articular surface with different grades.
DNA content
Cartilage explants were incubated overnight at 60 °C in 1 mL of papain digest solution [calcium- and magnesium-free PBS containing 1% papain suspension (Sigma, St Louis, MO), 5 mM cysteine, and 10 mM EDTA, pH 7.4]. The following day, aliquots of papain-digested cartilage were taken for DNA analysis using the Hoechst dye method and read on a fluorometer. The DNA concentration was extrapolated from a standard curve produced using calf thymus DNA (Sigma).
Total GAG content
An adaptation of the DMMB assay was used to measure sGAG content (Burkhardt et al. 2001). In brief, 25 mg of 1,9-DMMB was dissolved in 5 mL of ethanol and taken up to 1 L in 0.2% sodium formate buffer (pH 3.5). Using a multipipettor, 150 µL of the dye solution was dispensed into wells of a 96-well plate that contained 50 µg of papain-digested samples. A standard curve was generated with shark cartilage CSC (Sigma) at final concentrations of 1–10 µg. The samples were read on a microplate reader at 630 nm.
Extraction of GAGs from cartilage samples
Sulfated GAGs were released from the core protein in papain-digested cartilage samples with a 1.0 M NaBH4 and 0.05 M NaOH solution at 45 °C for 16 h. The pH was adjusted to 5.0 using glacial acetic acid. Hydrophobic biomolecules were removed from released cartilage samples via Pepclean C18 spin columns. The column was equilibrated with five 30 µL volumes of 0.1% acetic acid solution. The released cartilage sample was applied to the column, allowing 10 min for hydrophobic materials to adsorb to the C18 reversed-phase packing material. Released glycans were eluted with three 30 µL volumes of 0.1% acetic acid solution and dried in vacuo. The sample was dissolved in 50 µL of water and precipitated in nine volumes of chilled ethanol. Cationic biomolecules were removed from the GAG pellet via strong anion exchange microspin columns. The column was first hydrated with five 200 µL volumes of water and equilibrated with two 100 µL volumes of 300 mM sodium phosphate buffer for at least 1 h. The column was rinsed with three 100 µL volumes of 50 mM sodium phosphate buffer (pH 3.5). The GAG pellet was dissolved in 20 µL of 50 mM sodium phosphate (pH 3.5) and applied to the column, allowing 15 min for it to adsorb. Cationic biomolecules were washed off with three 100 µL volumes of 50 mM sodium phosphate pH 3.5. The GAG mixture was then eluted with two 100 µL volumes of 1 M NaCl, dried in vacuo, and precipitated in four volumes of chilled ethanol.
Preparation of -unsaturated CS oligosaccharides
A 10% fraction of each cartilage sample was digested exhaustively with chondroitinase ABC (2.5 µL, 4 mU/µL), chondroitinase AC1 (5 µL, 2 mU/µL), and chondroitinase B (4 µL, 0.5 mU/µL) at 37 °C; disaccharide products were then derivatized with AMAC. The remaining 90% of each sample was partially digested using chondroitinases to an absorbance value (232 nm) equal to 0.091 for bovine samples and 0.023 for human samples and boiled for 1 min. This degree of digestion was found to reproducibly depolymerize 10 µg of each respective cartilage sample to 30% reaction completion. On the basis of the relative chromatographic peak areas, the percentage of cartilage sample that was tetrasaccharide was found to be 28.2% on average in juvenile bovine and 31.6% on average in adult human. Oligosaccharide analysis of tetrasaccharides and hexasaccharides from the same dermatan sulfate samples resulted in similar signature ion profiles and quantification values (Miller et al. 2006). Given the nonspecific nature of the chondroitinase enzymes used, the glycoforms of the tetrasaccharides are likely to reflect the overall CS chain composition. Partial depolymerization products were lyophilized for subsequent reductive amination with 2-AA.
AMAC derivatization
Derivatization with AMAC was performed following the procedure described by Militsopoulou et al. (2002). Briefly, to the lyophilized cartilage GAG disaccharide sample were added 5 µL 0.1 M AMAC solutions in acetic acid: DMSO (3:17, v/v) and 5 µL of a freshly prepared 1 M NaBH3CN solution in water. The mixture was vortexed for 3 min and was incubated at 45 °C for 4 h after which 10 µL of DMSO was added. Excess reagents were removed with cellulose microspin columns as described in Derivatization sample clean-up.
Derivatization of oligosaccharides with d0- and d4-2-AA
All oligosaccharides were derivatized with d0- or d4-2-AA according to the method of Bigge et al. (1995). Briefly, a dried CSA sample (10 µg) was dissolved in 10 µL of a reaction reagent containing d0-2AA in DMSO/glacial acetic acid (7:3) and 1.0 M sodium cyanoborohydride. All other partially depolymerized cartilage samples were derivatized using d4-2AA under the same conditions. The glycan solutions were then centrifuged for 3 min and incubated at 65 °C for 3 h. Excess reagents were removed with cellulose microspin columns as described in Derivatization sample clean-up.
Derivatization sample clean-up
The cellulose column was first hydrated with five 200 µL volumes of water, rinsed with five 200 µL volumes of 30% acetic acid solution, and then with three 200 µL volumes of acetonitrile. The 2-AA derivatized reaction mixture was applied to the column, allowing 15 min for it to adsorb to the cellulose. Excess reagents were washed off with three 200 µL volumes of acetonitrile, followed by two 200 µL volumes of 96% acetonitrile. The derivatized glycan was then eluted with two 100 µL volumes of water and dried.
LC/MS/MS analysis
Derivatized glycan samples were fractionated using high-performance size-exclusion chromatography (SEC) with online tandem MS detection (Hitchcock et al. 2006). Briefly, the column (Superdex Peptide 3.2/30, Amersham Biosciences, Piscataway, NJ) was equilibrated in 10% acetonitrile and 0.05 M ammonium formate solution at 40 µL/min, and the oligosaccharide mixture (10 µL) was injected with UV detection at 310 nm.
The HPLC system was connected to a Bruker Daltonics (Billerica, MA) Esquire 3000 QITMS equipped with a standard electrospray ion source. The spray voltage was set at 3 kV; capillary temperature was set to 250 °C; the nebulizer gas (He) was set to 10 psi and the drying gas (N2) was set to 5 L/min. The capillary exit was set to –50.9 V and the skimmer potential was set to –10 V so as to prevent in-source fragmentation. Sample introduction into the mass spectrometer was achieved by connecting the SEC column to the sample inlet of the QIT electrospray source with peekTM tubing (Upchurch Scientific Inc., Oak Harbor, WA). The HPLC flow was split prior to the sample inlet, allowing 10 µL/min into the mass spectrometer. All scans were acquired in the negative ion mode using the automated MSn feature of the ion trap. The instrument was set to cycle between the MS and MS/MS modes until the entire mixture had eluted through the column. The MS/MS scans were summed for the most abundant charge state for the CS oligosaccharides; this corresponded to one negative charge per sulfate group.
CE-LIF analysis
The AMAC-derivatized cartilage samples were analyzed using a Beckman Coulter (Fullerton, CA) P/ACE MDQ capillary electrophoresis instrument. The uncoated fused silica capillary tubing (50 µm ID, 60µcm total length) was regenerated with 0.1 M HCl, 0.1 M NaOH, and HPLC-grade water before each run and analyses were performed using 50 mM NaH2PO4 buffer, pH 3.5, at 30 kV using reverse polarity and detected using the AMAC fluorophore (
ex=485 nm, em=510 nm). Baseline separation of the CS/DS isomers and good reproducibility were obtained when fresh anode and cathode buffer solutions were used for each run. A tri-sulfated heparin disaccharide (HSIS) was used as an internal standard for each run because it has a migration time different from all the CS disaccharides.
Methods' analysis and statistical tests
The coefficient of variation (%CV) was calculated to determine the minimal differences between samples that were detectable with CE and LC-MS/MS methods. For that purpose, a "sample" was defined as a single cartilage explant. The standard deviation of three independently analyzed aliquots from each sample was expressed as a percent of the mean (variance). The %CV was then calculated as the mean variance for a set of samples and was used to define the range of values predicted by experimental variation for specific glycoform abundances. The measured abundances of glycoforms and disaccharides were compared by Pearson correlation (SigmaStat for Windows software, version 2.03, SPSS Inc., Chicago, IL).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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We thank Drs Thomas Thornhill and John Wright for providing discarded tissues, and Christopher Rashidifard for technical assistance. Funding from NIH grants P41 RR10888, R01 HL74197, and R03 AG023307 is gratefully acknowledged. Bruker Daltonics donated the Esquire 3000 ion trap mass spectrometer used in this work.
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Footnotes |
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None declared.
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Abbreviations |
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AMAC, 2-aminoacridone; CE-LIF, capillary electrophoresis-laser-induced fluorescence; CID, collision-induced dissociation; CS, chondroitin sulfate; CSA, chondroitin sulfate type A; CSB, chondroitin sulfate type B; CSC, chondroitin sulfate type C; DMMB, dimethylmethylene blue; ECM, extracellular matrix; ESI, electrospray ionization; GAG, glycosaminoglycan; HPLC, high-performance liquid chromatography; KS, keratan sulfate; LC/MS/MS, liquid chromatography-tandem mass spectrometry; MS, mass spectrometry; OA, osteoarthritis; PG, proteoglycan; SEC, size-exclusion chromatography; SLRP, small leucine-rich proteoglycan; 2-AA, 2-anthranilic acid
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