1. Top of page
  2. Abstract
  7. Acknowledgements


To identify potential molecular mediators and biomarkers for osteoarthritis (OA), through comparative proteomic analysis of articular cartilage tissue obtained from normal donors without OA (n = 7) and patients with OA (n = 7).


The proteomic analyses comprised extraction of soluble proteins from cartilage, separation of the protein mixtures by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by in-gel digestion, and subsequent nano-liquid chromatography–tandem mass spectrometry analysis in conjunction with a database search for protein identification and semiquantitation.


A total of 814 distinct proteins were identified with high confidence from 14 samples; 420 of these proteins were detected with ≥3 unique peptides in at least 4 samples from the same group. Using stringent criteria, 59 proteins were found to be differentially expressed in OA cartilage. Gene Ontology and Ingenuity pathway analysis tools were used to characterize these proteins into functional categories. One of the up-regulated proteins, HtrA1, a serine protease, was detected at high levels in cartilage.


Altered protein expression in the disease state is associated with many aspects of the pathogenesis of OA, such as increased proteolysis, lipid metabolism, immune response, and decreased signal transduction. To our knowledge, this is the first time that a large portion of these proteins and their expression patterns were identified in cartilage, thus providing new insights for finding novel pathologic mediators and biomarkers of OA.

Osteoarthritis (OA) is a prevalent joint disease characterized by irreversible erosion and destruction of articular cartilage and subchondral bone. OA is a progressive, degenerative joint disease that has a major impact on joint function and the patient's quality of life, due to chronic pain. Cartilage is a connective tissue consisting of a single cell type (the chondrocyte, comprising 2–5% of the tissue weight) embedded in a dense extracellular matrix (ECM; primarily type II collagen, anionic aggrecan molecules, and hyaluronic acid) (1–6). It is generally accepted that, at the molecular level, cartilage degeneration is characterized by a general failure of chondrocytes to maintain an appropriate balance between synthesis and degradation of ECM macromolecules. Anabolic factors, such as various growth factors, bone morphogenetic proteins, and enzyme inhibitors, promote ECM production, while catabolic factors, including proinflammatory mediators (i.e., cytokines), chondrocyte apoptosis, and degradative enzymes and their activators, lead to the detrimental turnover of the ECM (6).

Differential proteomics aims to identify global protein components in biologic systems and provide a snapshot of the protein quantity change in response to pathologic stress, disease progression, or drug treatment. Both 2-dimensional (2-D) gel and automated multidimensional high-performance liquid chromatography (HPLC) in combination with mass spectrometry (MS) have been used with success for global profiling of complex protein mixtures such as cell lysates, tissues, and body fluids (7–11). For relative quantitation, proteins or their corresponding peptides in samples used for comparison can be differentially labeled, either metabolically or chemically, with stable isotopes, and the intensity ratio of the corresponding isotope-labeled peptide pairs can be measured by MS (12–14).

Recently, the capability of this approach has been extended from labeled pairs to multiplex labeling, allowing simultaneous quantitation of protein abundance in >2 samples (15–17). Alternatively, several investigators have demonstrated the feasibility of label-free approaches based on the measurement and comparison of peptide ion intensities for global semiquantitation of proteins (8, 11, 18–23). Chelius et al (19) showed that peak areas of peptide precursor ions, as calculated using alternating MS full scans in LC–tandem MS (LC-MS/MS) experiments, were closely correlated with protein concentrations, even in complex proteomes such as human serum. More recently, our group and several other groups of investigators (8, 11, 21) demonstrated that the peptide total ion intensity of a protein, based on MS scanning in data-dependent LC-MS/MS experiments, can be used as a measure of its relative abundance across multiple samples, to identify differentially expressed proteins. More importantly, the results obtained using this label-free approach correlated well with those obtained by independent proteomic studies based on 2-D gel or enzyme-linked immunosorbent assay (8, 11, 24).

Several studies have been conducted to search for pathologic mediators and/or biomarkers of OA using proteomic characterization of synovial fluids, serum, cultured chondrocytes, and cartilage tissue (2, 5, 25–32). Hermansson et al (2) used 2-D gel and MS to analyze newly synthesized proteins secreted by human OA cartilage explants cultured in medium containing 35S-methionine/cysteine. Those studies revealed increased synthesis of type II collagen and activin A proteins in OA cartilage. Ruiz-Romero et al (5) described protein profiles of human articular chondrocytes isolated from the cartilage of normal individuals, from which 93 unique proteins were identified. However, due to inherent alterations to the microenvironment, proteomic profiling of cultured chondrocytes may not fully reflect the spectrum of cellular proteins made in the cells surrounded by cartilage matrix in situ.

More recently, Vincourt et al (31) reported a method for direct proteomic characterization of human articular cartilage using 2-D electrophoresis and matrix-assisted laser desorption ionization−time-of-flight MS, leading to the identification of 127 proteins with diverse functions. Garcia et al (32) also reported proteomic profiling of OA cartilage using 1-D sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and tandem MS, which resulted in the identification of ∼100 proteins. The majority of these proteins were located in the ECM, since their sample preparation protocol excluded chondrocytes. To date, differential proteomic profiling of articular cartilage tissue from healthy individuals and patients with OA has not been reported.

To better understand the molecular mechanism and identify pathologic mediators and biomarkers for OA, we carried out a differential proteomic study to identify and semiquantitate proteins in articular cartilage tissue obtained from 7 patients with OA and 7 normal individuals. Our sample preparation protocol was designed to extract both extracellular and intracellular proteins from cartilage depleted of highly abundant matrix proteins such as collagens and aggrecan, thus allowing detection of many functionally important proteins that are usually less abundant in articular cartilage. A total of 814 distinct proteins were identified with 2 or more unique peptides, among which 59 proteins, including several that are implicated in OA pathology (such as matrix metalloproteinases [MMPs]), were found to be present at significantly different levels in OA cartilage compared with normal cartilage. The proteomic analysis also revealed several proteins that had not been previously reported to be associated with the pathology of OA.


  1. Top of page
  2. Abstract
  7. Acknowledgements

Extraction of soluble proteins from human cartilage.

Specimens of normal cartilage obtained from tissue donors with no history of joint disease were received within 36 hours following autopsies (performed by National Disease Research Interchange, Philadelphia, PA) and examined macroscopically to ensure that the cartilage specimens had smooth, intact surfaces without OA-like lesions. Specimens of OA cartilage with visible lesions were obtained from patients (ages 50–82 years) undergoing knee replacement surgery at New England Baptist Hospital, Boston, MA. These specimens were stored on ice for ∼36 hours before being processed for protein extraction.

Within this time frame, the majority (>90%) of chondrocytes were still alive, because the specimens were kept in Dulbecco's modified Eagle's medium–Ham's F-12 medium on ice. Applicable regulations and guidelines regarding patient consent and donor confidentiality were followed. Upon receipt, cartilage slices were carefully dissected and separated from other bony, bloody, or fibrous pieces. Cartilage tissue (1.2 gm wet weight) from each study subject was minced into ∼2 × 2 × 2–mm pieces and extracted for 4 days at 4°C with 12 ml of extraction buffer (4M guanidine HCl, 50 mM sodium acetate [pH 5.8]) containing a proteinase inhibitor cocktail (Roche, Grenzach, Germany). The resulting mixture was centrifuged at 3,000 revolutions per minute for 5 minutes at 4°C to remove the insoluble matrix (mostly collagen).

Depletion of aggrecan by CsCl gradient ultracentrifugation.

Solid CsCl was added to cartilage extracts to achieve a final density of 1.45 gm/ml, and the mixture was ultracentrifuged at 100,000g for 72 hours at 4°C. Three fractions of equal volume were collected, from top to bottom, for each tube. Essentially all of the sulfated proteoglycan (mainly aggrecan) fractionated in the bottom third of the tube, as measured by dimethylmethylene blue assay. Nonaggrecan cartilage proteins were found exclusively in the top fraction, as confirmed using SDS-PAGE gels. The top fraction was collected and desalted by dialysis at 4°C against 20 mM (pH 8.2) Tris HCl buffer containing proteinase inhibitor cocktail, using Slide-A-Lyzer 7K MWCO dialysis cassettes (Pierce, Rockford, IL). The concentration of the extracted proteins was measured by bicinchoninic acid assay (Pierce), following the manufacturer's protocol.

SDS-PAGE separation and in-gel tryptic digestion.

Each sample (containing 110 μg protein) was concentrated in a vacuum concentrator (SpeedVac; Thermo, San Jose, CA) to a final volume of ∼40 μl. The resulting solution was mixed with 5× SDS loading buffer. Proteins were reduced with 20 mM dithiothreitol at 90°C for 5 minutes and alkylated with 50 mM iodoacetamide at ambient temperature in the dark for 20 minutes. Samples were then loaded onto a 5-well 10–20% tricine mini-gel (Invitrogen, Carlsbad, CA). After staining with Coomassie blue, each gel lane was horizontally divided into 29 slices. Each slice was then minced into 1 × 1–mm pieces and subsequently subjected to in-gel digestion with sequencing-grade modified trypsin (0.5 μg/gel slice; Promega, Madison, WI) in a digestion robot (DigestPro; AbiMed Analysen-Technik, Langenfeld, Germany) for 18 hours. The tryptic digests were concentrated in a SpeedVac to a final volume of 30 μl, prior to MS analysis.


Tryptic digests of proteins were analyzed with an automated nanoLC-MS/MS system, using a Famos autosampler (LC Packings, San Francisco, CA) and an 1100 HPLC binary pump (Agilent, Wilmington, DE) coupled to an LTQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with a nanospray ionization source. Ten microliters of the digest solution was injected onto a reverse-phase PicoFrit column (New Objective, Woburn, MA) packed with Magic C18 media (5-μm particle, 200-angstrom pore size, 75 μm × 10 cm). Peptides were eluted at a flow rate of 0.2 μl/minute, using a 90-minute linear gradient from 2% to 55% B (mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 90% acetonitrile and 0.1% formic acid). The spray voltage was 1.8 kV, the heated capillary temperature was maintained at 180°C, and the collision energy for MS/MS was set at 35 units.

Automated data-dependent MS analysis was carried out using the dynamic exclusion feature built into the MS acquisition software (Xcalibur 1.3; Thermo Finnigan). Each MS full scan (mass/charge [m/z] 350–1,600) was followed by MS/MS acquisition of the 4 most intense precursor ions detected in the prior MS scan, to obtain as many collisionally induced dissociation spectra as possible.

Protein identification and annotation.

SpectrumMill version 3.1 software (Agilent) was used for database searches and for data processing. The mass spectrometric raw data were searched against the human subset of the National Center for Biotechnology Information (NCBI) nonredundant protein database (135,279 protein entries, updated as of July 2005). Search parameters included a static modification on cysteine residues (carbamidomethylation), signal-to-noise ratio >25, sequence tag length >1, precursor ion mass range 600–3,500 daltons, retention time 0–135 minutes, 70% minimum matched peak intensity ± 2.5-dalton tolerance on precursor ions and ± 0.7-dalton tolerance on product ions, 1 missed tryptic cleavage, and electrospray ionization trap scoring parameters as defined by the searching algorithm. Scans for the precursor ion m/z ± 0.7 dalton within a ±45-second time window were merged.

All of the database search results were further validated by applying the designated protein and peptide scores as well as the following user-defined criteria: for the protein validation mode, protein score >20, peptide scored percent intensity (SPI) >70% for all charge states, peptide score >7 for peptide charge +1, peptide score >8 for peptide charge +2, and peptide score >9 for peptide charges ≥+3; for the peptide validation mode, peptide SPI >70%, and peptide score >13 for all charge states. The search criteria used here would result in a false-positive rate of <4%, as previously described (8). The proteins identified from individual samples were reassembled to generate a summary that displays the results for all 14 samples. To address the database redundancy issue, proteins that share common peptides were grouped together and displayed as a single protein group in “Protein Centric Columns” mode, as described in the SpectrumMill search engine. Within any given protein group, the protein with the highest score was selected as the most likely correct search result.

To semiquantitate the protein relative abundance in multiple samples, the peak area of the extracted ion chromatograms for each peptide precursor ion in the full scan was calculated in the region ±1.4 m/z and ±75 scans, using SpectrumMill. An individual protein's abundance was then calculated as the sum of the total ion current (TIC) values measured for all peptide precursor ions derived from that protein. Thus, the relative concentration of each protein was determined by comparing total MS intensities of all identified peptides from that protein in 1 sample versus those from other samples. The annotation of protein cellular localization and biologic function was performed using Ingenuity ( and Gene Ontology (GO; software.

Selection of differentially expressed proteins.

The TIC for all peptides identified for a specific protein was used as a measure of relative abundance of that protein in the sample. Differential expression is based on the comparison of the mean TIC values for protein in OA and normal samples. Only proteins identified from ≥3 unique peptides were qualified for selection. A protein was considered to be differentially expressed if it was identified in ≥4 samples in at least 1 group (normal or OA) and the change in its mean TIC was >5-fold in either direction, from normal to OA tissue or from OA to normal tissue. The significance of such changes was measured by Student's t-tests of log10-transformed TIC values. The false discovery rate (FDR) adjustment to the resulting raw P values was calculated using the Benjamini-Hochberg procedure (33).


  1. Top of page
  2. Abstract
  7. Acknowledgements

Sample processing.

A reproducible procedure to remove highly abundant matrix macromolecules is critical to successful proteomic analysis of physiologically relevant proteins that may be present at very low levels. Following dissection and washing to remove extraneous proteins, cartilage specimens were extracted using 4M guanidine HCl in the presence of a proteinase inhibitor cocktail. Extraction using 4M guanidine HCl had been shown to be able to solubilize both the cellular and matrix proteins from cartilage (34). Collagens (primarily type II collagen), the most abundant cartilage matrix proteins, are insoluble under these conditions and were removed by centrifugation.

The cartilage extracts were further fractionated by CsCl gradient centrifugation, and the bulk of the aggrecan, the second most abundant protein component of cartilage, was subsequently separated from other cartilage proteins. SDS-PAGE analysis of the fractions from the CsCl gradients demonstrated that all of the cartilage proteins were accumulated in the top one-third fraction, while few or no proteins were detected in other fractions (data not shown). Thus, only the top one-third fraction (lowest buoyancy density) was processed for proteomic analysis. Cartilage sample information (donor age and sex) as well as the concentration of extracted cartilage protein from each study subject are listed in Supplementary Table 1 (available online at To compare relative protein expression in different samples, an equivalent amount of proteins from each sample (110 μg) was analyzed. As shown in the typical SDS-PAGE gel in Figure 1, the expression pattern of the abundant proteins in OA and normal samples appeared similar.

thumbnail image

Figure 1. Representative sodium dodecyl sulfate–polyacrylamide gel electrophoresis images of the extracted cartilage proteins in osteoarthritis (OA) and normal control (N) samples. An equivalent amount of the proteins (110 μg) was loaded for each sample.

Download figure to PowerPoint

Protein identification.

The number of proteins identified by LC-MS/MS in conjunction with database searching against human proteins in the NCBI nonredundant database is shown in Supplementary Table 1. Supplementary Tables 2 and 4 (available online at summarize the detailed information for each of the assigned proteins. There was no significant bias in the number of proteins identified in the OA or normal groups. A total of 814 distinct proteins representing a wide spectrum of functional classes were identified with high confidence from 14 cartilage samples (see Supplementary Table 2. Supplementary Tableq 4 lists an additional 400 proteins that were identified in this study, as assigned with a single unique peptide; however, these 400 proteins are not discussed in this report). Many of the identified proteins have not been previously reported to be components of articular cartilage. The cellular localization and molecular function of the identified proteins were categorized by GO and are shown in Figure 2. Both intracellular and extracellular proteins with various functions were identified. As expected, ECM proteins such as fibronectin, cartilage oligomeric matrix protein, thrombospondin 1, cartilage intermediate-layer protein, and decorin were among the most abundant, as measured by the sums of TIC values of their corresponding peptides. The protein list also contains several proteases and their inhibitors, as well as cytokines, kinases, and other signaling molecules that are usually present in very low amounts in cartilage.

thumbnail image

Figure 2. Gene Ontology annotation of the 814 distinct proteins identified from 14 cartilage samples. Results were obtained from DAVID Bioinformatics Resources at (47). A, Distribution of the identified proteins in various subcellular localizations. B, Distribution of identified proteins implicated in various molecular functions.

Download figure to PowerPoint

Selection of differentially expressed proteins.

To evaluate the relative changes in the levels of protein expression, a variety of criteria were applied, as follows: 1) the protein must be identified from ≥3 unique peptides; 2) the protein must be identified in ≥4 samples in at least 1 group; 3) the difference in the mean TIC values in 2 groups must be ≥5-fold (if the protein was not detected in a sample, a TIC value of 104, approximately equivalent to the noise level of the ion trap instrument, was arbitrarily assigned); and 4) FDR-adjusted (33) P values of ≤0.10 by t-test (corresponding to raw P values less than 0.03) were applied to further narrow down the list of differentially expressed proteins. Among the 814 unique proteins, 595 were identified from ≥3 peptides, and 420 of these 595 proteins were identified in ≥4 samples from at least 1 group (see Supplementary Table 3, available online at One hundred eighty-three of these proteins exhibited a mean TIC change of ≥5-fold in either direction. However, only 59 met the criteria after applying the FDR-adjusted P value cutoff (Table 1). The changes in these 59 proteins between normal and OA samples are also shown in the clustergram in Supplementary Figure 1 (online at For example, HtrA serine protease 11 (HtrA1; also called PRSS11), the most abundant protease in cartilage, was identified in all normal and OA samples from a total of 29 unique peptides (Table 1). Its expression in OA cartilage was ∼8-fold higher than that in normal cartilage (mean TIC1.8E + 09 versus 2.3E + 08).

Table 1. Detailed information on the 59 differentially expressed proteins in normal and osteoarthritis (OA) cartilage samples*
Protein nameAccession no.No. of unique peptidesLog, normal meanLog, OA meanFold changeFDR
  • *

    These proteins were selected from the 814 proteins identified from the 14 samples. The following criteria were applied: the protein must be identified with ≥3 unique peptides; the protein must be identified in ≥4 samples in at least 1 group; the difference of mean total ion currents in the normal and OA groups must be ≥5-fold; and false discovery rate (FDR)–adjusted t-test P values must be less than or equal to 0.10 (corresponding to raw P values of less than 0.03).

Thrombospondin 1 precursor40317626909.4688.553−8.20.041
Fibrinogen α-chain isoform11761629446.3549.086539.70.030
Apolipoprotein A-IV37499461395.9198.503383.20.044
Fibrinogen β-chain7924018356.7708.74594.30.030
Preapolipoprotein E178851337.7448.6828.70.030
Apolipoprotein A-I37499465326.1318.541257.30.030
Serine protease 114506141298.2249.1338.10.030
Fibrinogen, γ-chain isoform11761633255.9158.687590.80.030
EGF-containing fibulin-like ECM protein 115072400204.8327.566542.50.047
SMOC2 protein28839448176.1468.12795.70.087
Annexin VI, isoform 116877589167.2205.433−61.30.093
Hemoglobin β-chain4378804147.7648.6477.60.047
Complement component 4 binding protein4502503144.7417.144252.70.045
Matrix metalloproteinase 2 preproprotein11342666145.4567.554125.30.062
Complement factor H–related 513540563145.3217.651213.60.031
Nucleolin (protein C23)128841136.5354.514−105.00.073
Latent TGFβ-binding protein1082570137.1245.268−71.90.065
Tissue inhibitor of metalloproteinases 21517893126.0207.53132.40.087
Colα1 (III)4502951125.6527.49770.00.069
Hypothetical protein31873364127.2564.890−232.30.030
Anti-HBsAg immunoglobulin Fab κ-chain3721651117.8698.6405.90.073
Inter-alpha-trypsin inhibitor heavy-chain H2125000114.0007.4582,869.70.000
Apolipoprotein H (β2-glycoprotein I)32165624105.4937.840222.70.064
Similar to 60S acidic ribosomal protein P255635097105.6617.15731.30.094
F-box only protein 215812198107.4974.962−343.00.030
Complement factor B14124934104.7496.756101.60.065
Ig λ light-chain VLJ region2166950797.3568.0735.20.073
Coagulation factor II3080211594.0006.313205.80.030
Ig α-1 heavy-chain constant region18474994.0006.540347.10.030
Aldehyde reductase3058284586.6454.444−158.80.041
Histidine-rich glycoprotein450448974.2496.442156.00.047
Transmembrane protein 43718332175.2806.88139.90.093
Complement 9225812874.0006.388244.50.030
Glutathione S-transferase M2450417566.3174.380−86.50.039
D-dopachrome tautomerase3058277966.6994.955−55.40.030
Inositol(myo)-1 (or 4)-monophosphatase 1503178966.0234.000−105.40.030
H1 histone family, member X517444966.8684.364−319.20.003
Apolipoprotein A-II3058241164.4946.25157.20.047
Unknown (protein for IMAGE:4901992)3035394366.9214.957−92.20.094
Acrosomal serine protease inhibitor1319576964.0006.151141.70.030
TGFβ1-binding protein10794564.0006.453283.70.030
Proteasome β1 subunit1265347356.3034.257−111.40.030
MRCL3 protein1674104355.3186.92340.30.091
α1 acid glycoprotein 1119720944.0005.85771.90.073
Cystatin C3058251744.0005.58138.10.082
Acid phosphatase 1 isoform c475771444.3575.94838.90.090
Similar to fatty acid–binding protein, adipocyte5563085844.0005.73854.70.081
Small inducible cytokine B101501209935.5714.267−20.10.087
Branched-chain aminotransferase 1, cytosolic3817628735.8204.351−29.50.093
Transforming growth factor β11086387335.2104.000−16.20.073

The differentially expressed proteins shown in Table 1 represented a wide range of biologic categories, including major serum proteins such as apolipoproteins, hemopexin, fibrinogen, complement components; degradative proteases and protease inhibitors such as HtrA1, MMP/tissue inhibitor of metalloproteinases (TIMP), acrosomal serine protease inhibitor, and cystatin C; cartilage anabolic effectors such as different forms of transforming growth factor β (TGFβ) and TGFβ binding proteins; extracellular matrix proteins such as thrombospondin 1; as well as several interesting proteins whose properties and functions in OA pathophysiology have not been well characterized, such as fibulin 3, osteonectin, secreted modular calcium-binding protein 2, vitrin, and tenomodulin.

Bioinformatics characterization of cartilage proteins.

The proteins identified in cartilage tissue were analyzed by a variety of bioinformatics tools. Based on the TIC values of the 420 proteins (see Supplementary Table 3) identified in each experimental sample, the 14 cartilage samples could be separated into 2 distinct groups of OA and normal samples (Figure 3). This result demonstrated that OA cartilage samples have distinct protein expression profiles that can be distinguished from the protein profile of normal cartilage. Pathway analysis using tools provided by Ingenuity ( showed that the 59 differentially expressed proteins in OA (those with an FDR-adjusted P value of less than or equal to 0.10) cover a broad range of protein functional classes (Figure 4). A significant portion of proteins were involved in tissue development (16 of 59), cell–cell signaling and interaction (17 of 59), lipid/carbohydrate metabolism (18 of 59), molecular transport (13 of 59), cellular movement (12 of 59), cell death (7 of 59), and immune response (8 of 59).

thumbnail image

Figure 3. Sample correlation using the total ion current values from 420 selected proteins, each of which was detected from ≥3 unique peptides in at least 4 samples/group. The color scale ranges from 0 (no correlation) to 1 (self–self correlation). OA = osteoarthritis; N = normal.

Download figure to PowerPoint

thumbnail image

Figure 4. Ingenuity pathway analysis results, showing functional categories of the 59 differentially expressed proteins in osteoarthritis (OA) versus normal articular cartilage. The proteins are displayed according to physiologic function. The solid and open bars represent proteins that are increased or decreased in OA cartilage and normal articular cartilage, respectively. The y-axis represents the statistical significance of these changes. The numbers above the bars show the number of increased/decreased proteins involved in each functional category.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  7. Acknowledgements

In this study, we performed differential proteomics for the analysis of proteins in human articular cartilage, to elucidate changes in protein expression in patients with OA. The unique features of our study are as follows. First, this is, to our knowledge, the first application of comparative proteomics to cartilage tissue (as opposed to cultured chondrocytes) from normal individuals as well as patients with OA. Second, a total of 814 distinct proteins were identified, and a large proportion of these proteins and their expression changes was identified for the first time from cartilage tissue, thus providing a comprehensive cartilage proteome database that is essential in understanding the molecular mechanisms underlying OA. Third, 59 proteins displayed significant differences in abundance in association with OA. The functions of the proteins with significantly different abundance in OA versus normal articular cartilage may be associated with the pathogenesis of OA, such as proteolysis, lipid metabolism, immune response, cell death, and signal transduction.

A major obstacle in applying proteomics to cartilage tissue is the heterogeneous nature of cartilage proteins. Articular cartilage contains mainly ECM proteins, of which type II collagen and aggrecan comprise >90% of the total dry mass, whereas chondrocytes, the only cell type in cartilage, account for <5% of the total cartilage volume. As a result, the separation of crosslinked collagen and highly sulfated proteoglycan aggrecan was critical to the overall success of the strategy. In fact, neither collagens nor aggrecan was present as predominant cartilage proteins in our proteomic analyses.

Proteomic analysis of human articular cartilage demonstrated variability in the abundance of many proteins between different individuals, as indicated by highly variable TIC values for an individual protein in different samples. The criteria for selecting differential proteins included not only the confidence of the protein identification and the relative TIC changes but also the occurrence frequencies of the proteins within a group. These stringent criteria ensured that a selected protein was truly present in a majority of samples within a group, and that the altered abundance was not random. To facilitate the data processing, undetected (“absent”) proteins were arbitrarily assigned a TIC value of 104, which was approximately equivalent to the instrument noise level and significantly lower than most of the TIC values observed in the data set. Protein expression changes from normal to OA groups were assessed by the logarithmic difference of their mean TIC values, and the significance of such changes was assessed. The P value for the mean TIC change for each protein was calculated, and only those with an FDR-adjusted P value less than or equal to 0.1 were selected, to ensure that the changes in expression for individual proteins between the normal and OA groups were truly significant. This resulted in 59 proteins that were significantly changed (Table 1).

The etiology and pathophysiology of OA disease are complex and only partially understood. However, it is generally accepted that an imbalance between anabolic and catabolic pathways in matrix turnover contributes significantly to the mechanism (1, 3, 4, 6). The increased synthesis of degradative enzymes such as MMP and ADAMTS may largely account for the unbalanced breakdown of the cartilage ECM. Several cartilage proteases and protease inhibitors were identified in the current study. Among these, the levels of HtrA1, TIMP-2, MMP-2, cystatin C, and acrosomal serine protease inhibitor were all significantly elevated in OA cartilage, while the levels of many others were also increased, but to a lesser extent (Table 1 and Supplementary Table 3). HtrA1 was the most abundant protease identified in cartilage tissue, as estimated by the TIC value generated by unique peptides. This result confirmed previous findings reported by Hu et al (35) and Grau et al (36), showing elevated expression of HtrA1 at the transcriptional level in OA articular cartilage, and elevated levels of HtrA1 protein (∼7-fold above normal) in synovial fluids obtained from patients with OA.

In vitro studies, including those by our group, have shown that HtrA1 is able to digest several cartilage matrix proteins, including aggrecan, decorin, fibromodulin, and soluble type II collagen (37), and to inhibit TGFβ-induced matrix synthesis by chondrocytes (Yang Z, et al: unpublished observations). Chondrocytes also synthesize a variety of MMPs, including collagenases (MMP-1, MMP-8, and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysin 1 (MMP-3), and membrane-type MMPs. MMP-1, MMP-2, and MMP-3 were identified in this study, but only MMP-2 was altered in quantity in OA cartilage. Cysteine proteinases (such as cathepsins B, H, K, L, and S) represent another important enzyme family that might play a role in cartilage matrix destruction. Several cathepsins, particularly cathepsin K, have been shown to participate in OA cartilage degradation (38–40). Cystatin C, a potent extracellular inhibitor of cysteine proteinases, has also been linked to arthritis. Results from transcriptional profiling of cysteine proteinases and their inhibitors in a transgenic mouse model of OA showed that up-regulation of cathepsin K, but not cystatin expression, coincided with the onset of articular cartilage damage (41). However, our data strongly indicated that the level of cystatin C is increased in human OA cartilage.

The levels of several plasma proteins such as apolipoproteins (Apo A-I, II, IV, and Apo H) are significantly elevated in OA cartilage. The functional role of lipoproteins and their receptors in OA and other joint diseases has been described in the literature (42, 43). The observed up-regulation of these proteins in OA may also imply their involvement in disease mechanism that has not yet been fully investigated.

Finally, another of the interesting protein families identified in cartilage tissue comprised the fibulins. Fibulins are ECM proteins (44), and 6 different family members have been reported. All are characterized by a unique C-terminal fibulin-type module and several repeated epidermal growth factor–like domains. We identified 3 different fibulin proteins in cartilage: fibulin 1D, fibulin 2, and fibulin 3 (Figure 5). Fibulin 2 was identified in only 2 OA samples and 1 normal sample, at very low levels. In contrast, fibulin 1D and fibulin 3 were identified with strong abundance and were primarily observed in gel bands consistent with their intact molecular masses. The expression of fibulin 1D was slightly elevated in OA cartilage (mean TIC3.1E + 08 in OA cartilage versus 1.2E + 08 in normal cartilage), whereas expression of fibulin 3 was strongly increased in OA cartilage compared with normal cartilage (mean TIC3.6E + 08 versus 3.9E + 06).

thumbnail image

Figure 5. Expression profiles of fibulin family members (fibulin 2, fibulin 3, and fibulin 1D) in normal and osteoarthritis (OA) cartilage tissue. TIC = total ion current.

Download figure to PowerPoint

Fibulin 1D binds to proteoglycans and was recently reported as being a cofactor of ADAMTS-1, an inhibitor of angiogenesis and a mediator of proteolytic cleavage of aggrecan. When colocalized in vivo with ADAMTS-1, fibulin 1D was shown to enhance the capacity of ADAMTS-1 to cleave aggrecan (45). A recent study demonstrated that fibulin 3 is a binding partner of TIMP-3, which is a known inhibitor of MMPs and aggrecanases (46). Collectively, such evidence indicates that the fibulin family might play an important role in cartilage metabolism.

To our knowledge, the current study is the first comprehensive proteomic analysis of soluble proteins extracted from normal and OA cartilage. It provides the most complete catalog of the proteins involved in cellular metabolism and organization, as well as proteins participating in the regulation of ECM synthesis and turnover of articular cartilage. Although the present findings are based on a limited number of study subjects, the results, from the protein level, clearly reflect the complex pathophysiology of OA. This study provides new insights for identifying novel OA mediators that can be potential therapeutic targets or biomarkers for OA.


  1. Top of page
  2. Abstract
  7. Acknowledgements

Dr. Yang had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Wu, Bemis, Qiu, Flannery, Yang.

Acquisition of data. Wu, Bemis, Wang, Yang.

Analysis and interpretation of data. Wu, Liu, Bemis, Morris, Flannery, Yang.

Manuscript preparation. Wu, Liu, Flannery, Yang.

Statistical analysis. Wu, Liu, Bemis, Yang.


  1. Top of page
  2. Abstract
  7. Acknowledgements

We thank our colleagues Edward LaVallie, Lisa A. Collins-Racie, Maya Arai, Katy Georgiadis, Manas Majumdar, Sonya Glasson, Vishnu Daesety, Priya Chocklingam, and Aled Jones for constructive comments, and Ioannis Moutsatsos and Patrick Cody for software support. We also are grateful to the New England Baptist Hospital (Boston, MA) and the National Disease Research Interchange (Philadelphia, PA) for providing cartilage tissue.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  • 1
    Berenbaum F. Signaling transduction: target in osteoarthritis. Curr Opin Rheumatol 2004; 16: 61622.
  • 2
    Hermansson M, Sawaji Y, Bolton M, Alexander S, Wallace A, Begum S, et al. Proteomic analysis of articular cartilage shows increased type II collagen synthesis in osteoarthritis and expression of inhibin βA (activin A), a regulatory molecule for chondrocytes. J Biol Chem 2004; 279: 4351421.
  • 3
    Malemud CJ. Fundamental pathways in osteoarthritis: an overview [review]. Front Biosci 1999; 4: D65961.
  • 4
    Malemud CJ, Islam N, Haqqi TM. Pathophysiological mechanisms in osteoarthritis lead to novel therapeutic strategies [review]. Cells Tissues Organs 2003; 174: 3448.
  • 5
    Ruiz-Romero C, Lopez-Armada MJ, Blanco FJ. Proteomic characterization of human normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases. Proteomics 2005; 5: 304859.
  • 6
    Smith RL. Degradative enzymes in osteoarthritis. Front Biosci 1999; 4: D70412.
  • 7
    Adkins JN, Varnum SM, Auberry KJ, Moore RJ, Angell NH, Smith RD, et al. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol Cell Proteomics 2002; 1: 94755.
  • 8
    Liao H, Wu J, Kuhn E, Chin W, Chang B, Jones MD, et al. Use of mass spectrometry to identify protein biomarkers of disease severity in the synovial fluid and serum of patients with rheumatoid arthritis. Arthritis Rheum 2004; 50: 3792803.
  • 9
    Washburn MP, Ulaszek R, Deciu C, Schieltz DM, Yates JR III. Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Anal Chem 2002; 74: 16507.
  • 10
    Washburn MP, Wolters D, Yates JR III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001; 19: 2427.
  • 11
    Wu J, Kobayashi M, Sousa EA, Liu W, Cai J, Goldman SJ, et al. Differential proteomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental antigen challenge. Mol Cell Proteomics 2005; 4: 125164.
  • 12
    Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 1999; 17: 9949.
  • 13
    Gygi SP, Rist B, Griffin TJ, Eng J, Aebersold R. Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags. J Proteome Res 2002; 1: 4754.
  • 14
    Li J, Steen H, Gygi SP. Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response. Mol Cell Proteomics 2003; 2: 1198204.
  • 15
    Cong YS, Fan E, Wang E. Simultaneous proteomic profiling of four different growth states of human fibroblasts, using amine-reactive isobaric tagging reagents and tandem mass spectrometry. Mech Ageing Dev 2006; 127: 33243.
  • 16
    Hardt M, Witkowska HE, Webb S, Thomas LR, Dixon SE, Hall SC, et al. Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents. Anal Chem 2005; 77: 494754.
  • 17
    Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 2004; 3: 115469.
  • 18
    Bondarenko PV, Chelius D, Shaler TA. Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry. Anal Chem 2002; 74: 47419.
  • 19
    Chelius D, Zhang T, Wang G, Shen RF. Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry. Anal Chem 2003; 75: 665865.
  • 20
    Forner F, Foster LJ, Campanaro S, Valle G, Mann M. Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver. Mol Cell Proteomics 2006; 5: 60819.
  • 21
    Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, et al. Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 2005; 4: 1487502.
  • 22
    Ono M, Shitashige M, Honda K, Isobe T, Kuwabara H, Matsuzuki H, et al. Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry. Mol Cell Proteomics 2006; 5: 133847.
  • 23
    Wang W, Zhou H, Lin H, Roy S, Shaler TA, Hill LR, et al. Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal Chem 2003; 75: 481826.
  • 24
    Drynda S, Ringel B, Kekow M, Kuhne C, Drynda A, Glocker MO, et al. Proteome analysis reveals disease-associated marker proteins to differentiate RA patients from other inflammatory joint diseases with the potential to monitor anti-TNFα therapy. Pathol Res Pract 2004; 200: 16571.
  • 25
    Sinz A, Bantscheff M, Mikkat S, Ringel B, Drynda S, Kekow J, et al. Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. Electrophoresis 2002; 23: 344556.
  • 26
    Xiang Y, Sekine T, Nakamura H, Imajoh-Ohmi S, Fukuda H, Nishioka K, et al. Proteomic surveillance of autoimmunity in osteoarthritis: identification of triosephosphate isomerase as an autoantigen in patients with osteoarthritis. Arthritis Rheum 2004; 50: 151121.
  • 27
    Xiang Y, Sekine T, Nakamura H, Imajoh-Ohmi S, Fukuda H, Yudoh K, et al. Fibulin-4 is a target of autoimmunity predominantly in patients with osteoarthritis. J Immunol 2006; 176: 3196204.
  • 28
    Tilleman K, Van Beneden K, Dhondt A, Hoffman I, De Keyser F, Veys E, et al. Chronically inflamed synovium from spondyloarthropathy and rheumatoid arthritis investigated by protein expression profiling followed by tandem mass spectrometry. Proteomics 2005; 5: 224757.
  • 29
    Yamagiwa H, Sarkar G, Charlesworth MC, McCormick DJ, Bolander ME. Two-dimensional gel electrophoresis of synovial fluid: method for detecting candidate protein markers for osteoarthritis. J Orthop Sci 2003; 8: 48290.
  • 30
    De Ceuninck F, Marcheteau E, Berger S, Caliez A, Dumont V, Raes M, et al. Assessment of some tools for the characterization of the human osteoarthritic cartilage proteome. J Biomol Tech 2005; 16: 25665.
  • 31
    Vincourt JB, Lionneton F, Kratassiouk G, Guillemin F, Netter P, Mainard D, et al. Establishment of a reliable method for direct proteome characterization of human articular cartilage. Mol Cell Proteomics 2006; 5: 198495.
  • 32
    Garcia BA, Platt MD, Born TL, Shabanowitz J, Marcus NA, Hunt DF. Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry. Rapid Commun Mass Spectrom 2006; 20: 29993006.
  • 33
    Reiner A, Yekutieli D, Benjamini Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 2003; 19: 36875.
  • 34
    Hoemann CD, Sun J, Chrzanowski V, Buschmann MD. A multivalent assay to detect glycosaminoglycan, protein, collagen, RNA, and DNA content in milligram samples of cartilage or hydrogel-based repair cartilage. Anal Biochem 2002; 300: 110.
  • 35
    Hu SI, Carozza M, Klein M, Nantermet P, Luk D, Crowl RM. Human HtrA, an evolutionarily conserved serine protease identified as a differentially expressed gene product in osteoarthritic cartilage. J Biol Chem 1998; 273: 3440612.
  • 36
    Grau S, Baldi A, Bussani R, Tian X, Stefanescu R, Przybylski M, et al. Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc Natl Acad Sci U S A 2005; 102: 60216.
  • 37
    Tsuchiya A, Yano M, Tocharus J, Kojima H, Fukumoto M, Kawaichi M, et al. Expression of mouse HtrA1 serine protease in normal bone and cartilage and its upregulation in joint cartilage damaged by experimental arthritis. Bone 2005; 37: 32336.
  • 38
    Hou WS, Li W, Keyszer G, Weber E, Levy R, Klein MJ, et al. Comparison of cathepsins K and S expression within the rheumatoid and osteoarthritic synovium. Arthritis Rheum 2002; 46: 66374.
  • 39
    Konttinen YT, Mandelin J, Li TF, Salo J, Lassus J, Liljestrom M, et al. Acidic cysteine endoproteinase cathepsin K in the degeneration of the superficial articular hyaline cartilage in osteoarthritis. Arthritis Rheum 2002; 46: 95360.
  • 40
    Mandelin J, Hukkanen M, Li TF, Korhonen M, Liljestrom M, Sillat T, et al. Human osteoblasts produce cathepsin K. Bone 2006; 38: 76977.
  • 41
    Morko JP, Soderstrom M, Saamanen AM, Salminen HJ, Vuorio EI. Up regulation of cathepsin K expression in articular chondrocytes in a transgenic mouse model for osteoarthritis. Ann Rheum Dis 2004; 63: 64955.
  • 42
    Aspden RM, Scheven BA, Hutchison JD. Osteoarthritis as a systemic disorder including stromal cell differentiation and lipid metabolism. Lancet 2001; 357: 111820.
  • 43
    Kakinuma T, Yasuda T, Nakagawa T, Hiramitsu T, Akiyoshi M, Akagi M, et al. Lectin-like oxidized low-density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low-density lipoprotein in rheumatoid arthritis cartilage. Arthritis Rheum 2004; 50: 3495503.
  • 44
    Timpl R, Sasaki T, Kostka G, Chu ML. Fibulins: a versatile family of extracellular matrix proteins. Nat Rev Mol Cell Biol 2003; 4: 47989.
  • 45
    Lee NV, Rodriguez-Manzaneque JC, Thai SN, Twal WO, Luque A, Lyons KM, et al. Fibulin-1 acts as a cofactor for the matrix metalloprotease ADAMTS-1. J Biol Chem 2005; 280: 34796804.
  • 46
    Klenotic PA, Munier FL, Marmorstein LY, Anand-Apte B. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a binding partner of epithelial growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1): implications for macular degenerations. J Biol Chem 2004; 279: 3046973.
  • 47
    Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol 2003; 4: P3.