ARTHRITIS & RHEUMATISMVol. 56, No. 11, November 2007, pp 3675–3684DOI 10.1002/art.22876© 2007, American College of Rheumatology

Comparative Proteomic Characterization of Articular CartilageTissue From Normal Donors and Patients With Osteoarthritis

Jiang Wu, Wei Liu, Amanda Bemis, Eunice Wang, Yongchang Qiu, Elisabeth A. Morris,Carl R. Flannery, and Zhiyong Yang

Objective. To identify potential molecular media-tors and biomarkers for osteoarthritis (OA), throughcomparative proteomic analysis of articular cartilagetissue obtained from normal donors without OA (n � 7)and patients with OA (n � 7).

Methods. The proteomic analyses comprised ex-traction of soluble proteins from cartilage, separation ofthe protein mixtures by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by in-gel di-gestion, and subsequent nano-liquid chromatography–tandem mass spectrometry analysis in conjunction with adatabase search for protein identification and semi-quantitation.

Results. A total of 814 distinct proteins wereidentified with high confidence from 14 samples; 420 ofthese proteins were detected with >3 unique peptides inat least 4 samples from the same group. Using stringentcriteria, 59 proteins were found to be differentiallyexpressed in OA cartilage. Gene Ontology and Ingenuitypathway analysis tools were used to characterize theseproteins into functional categories. One of the up-regulated proteins, HtrA1, a serine protease, was de-tected at high levels in cartilage.

Conclusion. Altered protein expression in thedisease state is associated with many aspects of thepathogenesis of OA, such as increased proteolysis, lipidmetabolism, immune response, and decreased signal

transduction. To our knowledge, this is the first timethat a large portion of these proteins and their expres-sion patterns were identified in cartilage, thus providingnew insights for finding novel pathologic mediators andbiomarkers of OA.

Osteoarthritis (OA) is a prevalent joint diseasecharacterized by irreversible erosion and destruction ofarticular cartilage and subchondral bone. OA is a pro-gressive, degenerative joint disease that has a majorimpact on joint function and the patient’s quality of life,due to chronic pain. Cartilage is a connective tissueconsisting of a single cell type (the chondrocyte, com-prising 2–5% of the tissue weight) embedded in a denseextracellular 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 generalfailure of chondrocytes to maintain an appropriate bal-ance between synthesis and degradation of ECM macro-molecules. Anabolic factors, such as various growthfactors, bone morphogenetic proteins, and enzyme in-hibitors, promote ECM production, while catabolic fac-tors, including proinflammatory mediators (i.e., cyto-kines), chondrocyte apoptosis, and degradative enzymesand their activators, lead to the detrimental turnover ofthe ECM (6).

Differential proteomics aims to identify globalprotein components in biologic systems and provide asnapshot of the protein quantity change in response topathologic stress, disease progression, or drug treat-ment. Both 2-dimensional (2-D) gel and automatedmultidimensional high-performance liquid chromatog-raphy (HPLC) in combination with mass spectrometry(MS) have been used with success for global profiling ofcomplex protein mixtures such as cell lysates, tissues,and body fluids (7–11). For relative quantitation, pro-teins or their corresponding peptides in samples used for

Jiang Wu, PhD, Wei Liu, PhD, Amanda Bemis, BS, EuniceWang, MS, Yongchang Qiu, PhD, Elisabeth A. Morris, DMV, Carl R.Flannery, PhD, Zhiyong Yang, PhD: Wyeth Research, Cambridge,Massachusetts.

Drs. Wu, Liu, Qiu, Morris, Flannery, and Yang own stockand/or stock options in Wyeth.

Address correspondence and reprint requests to ZhiyongYang, PhD, Wyeth Research, 200 Cambridge Park Drive, Cambridge,MA 02140 (e-mail: zyyang@wyeth.com); or to Jiang Wu, PhD, WyethResearch, 200 Cambridge Park Drive, Cambridge, MA 02140 (e-mail:jxwu@wyeth.com).

Submitted for publication March 9, 2007; accepted in revisedform June 12, 2007.

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comparison can be differentially labeled, either metabol-ically or chemically, with stable isotopes, and the inten-sity ratio of the corresponding isotope-labeled peptidepairs can be measured by MS (12–14).

Recently, the capability of this approach has beenextended from labeled pairs to multiplex labeling, allow-ing simultaneous quantitation of protein abundance in�2 samples (15–17). Alternatively, several investigatorshave demonstrated the feasibility of label-free ap-proaches based on the measurement and comparison ofpeptide ion intensities for global semiquantitation ofproteins (8,11,18–23). Chelius et al (19) showed thatpeak areas of peptide precursor ions, as calculated usingalternating MS full scans in LC–tandem MS (LC-MS/MS) experiments, were closely correlated with proteinconcentrations, even in complex proteomes such ashuman serum. More recently, our group and severalother groups of investigators (8,11,21) demonstratedthat the peptide total ion intensity of a protein, based onMS scanning in data-dependent LC-MS/MS experi-ments, can be used as a measure of its relative abun-dance across multiple samples, to identify differentiallyexpressed proteins. More importantly, the results ob-tained using this label-free approach correlated well withthose obtained by independent proteomic studies basedon 2-D gel or enzyme-linked immunosorbent assay(8,11,24).

Several studies have been conducted to search forpathologic mediators and/or biomarkers of OA usingproteomic 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 analyzenewly synthesized proteins secreted by human OA car-tilage explants cultured in medium containing 35S-methionine/cysteine. Those studies revealed increasedsynthesis of type II collagen and activin A proteins inOA cartilage. Ruiz-Romero et al (5) described proteinprofiles of human articular chondrocytes isolated fromthe cartilage of normal individuals, from which 93unique proteins were identified. However, due to inher-ent alterations to the microenvironment, proteomic pro-filing of cultured chondrocytes may not fully reflect thespectrum of cellular proteins made in the cells sur-rounded by cartilage matrix in situ.

More recently, Vincourt et al (31) reported amethod for direct proteomic characterization of humanarticular cartilage using 2-D electrophoresis and matrix-assisted laser desorption ionization�time-of-flight MS,leading to the identification of 127 proteins with diversefunctions. Garcia et al (32) also reported proteomicprofiling of OA cartilage using 1-D sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and tandem MS, which resulted in the identifi-cation of �100 proteins. The majority of these proteinswere located in the ECM, since their sample preparationprotocol excluded chondrocytes. To date, differentialproteomic profiling of articular cartilage tissue fromhealthy individuals and patients with OA has not beenreported.

To better understand the molecular mechanismand identify pathologic mediators and biomarkers forOA, we carried out a differential proteomic study toidentify and semiquantitate proteins in articular carti-lage tissue obtained from 7 patients with OA and 7normal individuals. Our sample preparation protocolwas designed to extract both extracellular and intracell-ular proteins from cartilage depleted of highly abundantmatrix proteins such as collagens and aggrecan, thusallowing detection of many functionally important pro-teins that are usually less abundant in articular cartilage.A total of 814 distinct proteins were identified with 2 ormore unique peptides, among which 59 proteins, includ-ing several that are implicated in OA pathology (such asmatrix metalloproteinases [MMPs]), were found to bepresent at significantly different levels in OA cartilagecompared with normal cartilage. The proteomic analysisalso revealed several proteins that had not been previ-ously reported to be associated with the pathology ofOA.

PATIENTS AND METHODS

Extraction of soluble proteins from human cartilage.Specimens of normal cartilage obtained from tissue donorswith no history of joint disease were received within 36 hoursfollowing autopsies (performed by National Disease ResearchInterchange, Philadelphia, PA) and examined macroscopicallyto ensure that the cartilage specimens had smooth, intactsurfaces without OA-like lesions. Specimens of OA cartilagewith visible lesions were obtained from patients (ages 50–82years) undergoing knee replacement surgery at New EnglandBaptist Hospital, Boston, MA. These specimens were storedon ice for �36 hours before being processed for proteinextraction.

Within this time frame, the majority (�90%) of chon-drocytes were still alive, because the specimens were kept inDulbecco’s modified Eagle’s medium–Ham’s F-12 medium onice. Applicable regulations and guidelines regarding patientconsent and donor confidentiality were followed. Upon re-ceipt, cartilage slices were carefully dissected and separatedfrom other bony, bloody, or fibrous pieces. Cartilage tissue (1.2gm 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 ofextraction 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

3676 WU ET AL

3,000 revolutions per minute for 5 minutes at 4°C to removethe insoluble matrix (mostly collagen).

Depletion of aggrecan by CsCl gradient ultracentrifu-gation. Solid CsCl was added to cartilage extracts to achieve afinal density of 1.45 gm/ml, and the mixture was ultracentri-fuged at 100,000g for 72 hours at 4°C. Three fractions of equalvolume 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 bydimethylmethylene blue assay. Nonaggrecan cartilage proteinswere found exclusively in the top fraction, as confirmed usingSDS-PAGE gels. The top fraction was collected and desaltedby dialysis at 4°C against 20 mM (pH 8.2) Tris HCl buffercontaining proteinase inhibitor cocktail, using Slide-A-Lyzer7K MWCO dialysis cassettes (Pierce, Rockford, IL). Theconcentration of the extracted proteins was measured bybicinchoninic acid assay (Pierce), following the manufacturer’sprotocol.

SDS-PAGE separation and in-gel tryptic digestion.Each sample (containing 110 �g protein) was concentrated ina vacuum concentrator (SpeedVac; Thermo, San Jose, CA) toa final volume of �40 �l. The resulting solution was mixed with5� SDS loading buffer. Proteins were reduced with 20 mMdithiothreitol at 90°C for 5 minutes and alkylated with 50 mMiodoacetamide at ambient temperature in the dark for 20minutes. Samples were then loaded onto a 5-well 10–20%tricine mini-gel (Invitrogen, Carlsbad, CA). After staining withCoomassie blue, each gel lane was horizontally divided into 29slices. Each slice was then minced into 1 � 1–mm pieces andsubsequently subjected to in-gel digestion with sequencing-grade modified trypsin (0.5 �g/gel slice; Promega, Madison,WI) in a digestion robot (DigestPro; AbiMed Analysen-Technik, Langenfeld, Germany) for 18 hours. The trypticdigests were concentrated in a SpeedVac to a final volume of30 �l, prior to MS analysis.

NanoLC-MS/MS. Tryptic digests of proteins were an-alyzed with an automated nanoLC-MS/MS system, using aFamos autosampler (LC Packings, San Francisco, CA) and an1100 HPLC binary pump (Agilent, Wilmington, DE) coupledto an LTQ ion trap mass spectrometer (Thermo Finnigan, SanJose, CA) equipped with a nanospray ionization source. Tenmicroliters of the digest solution was injected onto a reverse-phase PicoFrit column (New Objective, Woburn, MA) packedwith Magic C18 media (5-�m particle, 200-angstrom pore size,75 �m � 10 cm). Peptides were eluted at a flow rate of 0.2�l/minute, using a 90-minute linear gradient from 2% to 55%B (mobile phase A: 0.1% formic acid aqueous solution; mobilephase B: 90% acetonitrile and 0.1% formic acid). The sprayvoltage was 1.8 kV, the heated capillary temperature wasmaintained at 180°C, and the collision energy for MS/MS wasset at 35 units.

Automated data-dependent MS analysis was carriedout using the dynamic exclusion feature built into the MSacquisition software (Xcalibur 1.3; Thermo Finnigan). EachMS full scan (mass/charge [m/z] 350–1,600) was followed byMS/MS acquisition of the 4 most intense precursor ionsdetected in the prior MS scan, to obtain as many collisionallyinduced dissociation spectra as possible.

Protein identification and annotation. SpectrumMillversion 3.1 software (Agilent) was used for database searchesand for data processing. The mass spectrometric raw data were

searched against the human subset of the National Center forBiotechnology Information (NCBI) nonredundant protein da-tabase (135,279 protein entries, updated as of July 2005).Search parameters included a static modification on cysteineresidues (carbamidomethylation), signal-to-noise ratio �25,sequence tag length �1, precursor ion mass range 600–3,500daltons, retention time 0–135 minutes, 70% minimum matchedpeak 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 definedby 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 vali-dated by applying the designated protein and peptide scores aswell as the following user-defined criteria: for the proteinvalidation mode, protein score �20, peptide scored percentintensity (SPI) �70% for all charge states, peptide score �7for peptide charge �1, peptide score �8 for peptide charge�2, and peptide score �9 for peptide charges ��3; for thepeptide validation mode, peptide SPI �70%, and peptidescore �13 for all charge states. The search criteria used herewould result in a false-positive rate of �4%, as previouslydescribed (8). The proteins identified from individual sampleswere reassembled to generate a summary that displays theresults for all 14 samples. To address the database redundancyissue, proteins that share common peptides were groupedtogether and displayed as a single protein group in “ProteinCentric Columns” mode, as described in the SpectrumMillsearch engine. Within any given protein group, the proteinwith the highest score was selected as the most likely correctsearch result.

To semiquantitate the protein relative abundance inmultiple samples, the peak area of the extracted ion chromato-grams for each peptide precursor ion in the full scan wascalculated in the region �1.4 m/z and �75 scans, usingSpectrumMill. An individual protein’s abundance was thencalculated as the sum of the total ion current (TIC) valuesmeasured for all peptide precursor ions derived from thatprotein. Thus, the relative concentration of each protein wasdetermined by comparing total MS intensities of all identifiedpeptides from that protein in 1 sample versus those from othersamples. The annotation of protein cellular localization andbiologic function was performed using Ingenuity (www.ingenuity.com) and Gene Ontology (GO; http://amigo.geneontology.org) software.

Selection of differentially expressed proteins. The TICfor all peptides identified for a specific protein was used as ameasure of relative abundance of that protein in the sample.Differential expression is based on the comparison of the meanTIC values for protein in OA and normal samples. Onlyproteins identified from �3 unique peptides were qualified forselection. A protein was considered to be differentially ex-pressed if it was identified in �4 samples in at least 1 group(normal or OA) and the change in its mean TIC was �5-foldin either direction, from normal to OA tissue or from OA tonormal tissue. The significance of such changes was measuredby Student’s t-tests of log10-transformed TIC values. The falsediscovery rate (FDR) adjustment to the resulting raw P valueswas calculated using the Benjamini-Hochberg procedure (33).

DIFFERENTIAL PROTEOMIC PROFILING OF HUMAN NORMAL AND OA CARTILAGE 3677

RESULTS

Sample processing. A reproducible procedure toremove highly abundant matrix macromolecules is crit-ical to successful proteomic analysis of physiologicallyrelevant proteins that may be present at very low levels.Following dissection and washing to remove extraneousproteins, cartilage specimens were extracted using 4Mguanidine HCl in the presence of a proteinase inhibitorcocktail. Extraction using 4M guanidine HCl had beenshown to be able to solubilize both the cellular andmatrix proteins from cartilage (34). Collagens (primarilytype II collagen), the most abundant cartilage matrixproteins, are insoluble under these conditions and wereremoved by centrifugation.

The cartilage extracts were further fractionatedby CsCl gradient centrifugation, and the bulk of theaggrecan, the second most abundant protein componentof cartilage, was subsequently separated from othercartilage proteins. SDS-PAGE analysis of the fractionsfrom the CsCl gradients demonstrated that all of thecartilage proteins were accumulated in the top one-thirdfraction, while few or no proteins were detected in otherfractions (data not shown). Thus, only the top one-thirdfraction (lowest buoyancy density) was processed forproteomic analysis. Cartilage sample information (donorage and sex) as well as the concentration of extractedcartilage protein from each study subject are listed inSupplementary Table 1 (available online at http://bioinfo.public.wyeth.com/WHB). To compare relativeprotein expression in different samples, an equivalentamount of proteins from each sample (110 �g) wasanalyzed. As shown in the typical SDS-PAGE gel inFigure 1, the expression pattern of the abundant pro-teins in OA and normal samples appeared similar.

Protein identification. The number of proteinsidentified by LC-MS/MS in conjunction with databasesearching against human proteins in the NCBI nonre-dundant database is shown in Supplementary Table 1.Supplementary Tables 2 and 4 (available online athttp://bioinfo.public.wyeth.com/WHB) summarize thedetailed information for each of the assigned proteins.There was no significant bias in the number of proteinsidentified in the OA or normal groups. A total of 814distinct proteins representing a wide spectrum of func-tional classes were identified with high confidence from14 cartilage samples (see Supplementary Table 2. Sup-plementary Table 4 lists an additional 400 proteins thatwere identified in this study, as assigned with a singleunique peptide; however, these 400 proteins are notdiscussed in this report). Many of the identified proteins

have not been previously reported to be components ofarticular cartilage. The cellular localization and molec-ular function of the identified proteins were categorizedby GO and are shown in Figure 2. Both intracellular andextracellular proteins with various functions were iden-tified. As expected, ECM proteins such as fibronectin,cartilage oligomeric matrix protein, thrombospondin 1,

Figure 2. Gene Ontology annotation of the 814 distinct proteinsidentified from 14 cartilage samples. Results were obtained fromDAVID Bioinformatics Resources at http://david.abcc.ncifcrf.gov/home.jsp (47). A, Distribution of the identified proteins in varioussubcellular localizations. B, Distribution of identified proteins impli-cated in various molecular functions.

Figure 1. Representative sodium dodecyl sulfate–polyacrylamide gelelectrophoresis images of the extracted cartilage proteins in osteoar-thritis (OA) and normal control (N) samples. An equivalent amount ofthe proteins (110 �g) was loaded for each sample.

3678 WU ET AL

Table 1. Detailed information on the 59 differentially expressed proteins in normal and osteoarthritis (OA) cartilage samples*

Protein name Accession no.No. of unique

peptidesLog, normal

meanLog, OA

meanFold

change FDR

Thrombospondin 1 precursor 40317626 90 9.468 8.553 �8.2 0.041Fibrinogen �-chain isoform 11761629 44 6.354 9.086 539.7 0.030Apolipoprotein A-IV 37499461 39 5.919 8.503 383.2 0.044Fibrinogen �-chain 7924018 35 6.770 8.745 94.3 0.030Preapolipoprotein E 178851 33 7.744 8.682 8.7 0.030Apolipoprotein A-I 37499465 32 6.131 8.541 257.3 0.030Serine protease 11 4506141 29 8.224 9.133 8.1 0.030VIT 37181801 28 8.289 5.401 �772.5 0.030Fibrinogen, �-chain isoform 11761633 25 5.915 8.687 590.8 0.030EGF-containing fibulin-like ECM protein 1 15072400 20 4.832 7.566 542.5 0.047SMOC2 protein 28839448 17 6.146 8.127 95.7 0.087Annexin VI, isoform 1 16877589 16 7.220 5.433 �61.3 0.093Hemoglobin �-chain 4378804 14 7.764 8.647 7.6 0.047KIAA0120 40788953 14 5.494 7.192 50.0 0.062Complement component 4 binding protein 4502503 14 4.741 7.144 252.7 0.045Matrix metalloproteinase 2 preproprotein 11342666 14 5.456 7.554 125.3 0.062Complement factor H–related 5 13540563 14 5.321 7.651 213.6 0.031Nucleolin (protein C23) 128841 13 6.535 4.514 �105.0 0.073Latent TGF�-binding protein 1082570 13 7.124 5.268 �71.9 0.065Tissue inhibitor of metalloproteinases 2 1517893 12 6.020 7.531 32.4 0.087Col�1 (III) 4502951 12 5.652 7.497 70.0 0.069Hypothetical protein 31873364 12 7.256 4.890 �232.3 0.030Anti-HBsAg immunoglobulin Fab �-chain 3721651 11 7.869 8.640 5.9 0.073PSME1 49456277 11 6.402 4.208 �156.3 0.030Inter-alpha-trypsin inhibitor heavy-chain H2 125000 11 4.000 7.458 2,869.7 0.000Apolipoprotein H (�2-glycoprotein I) 32165624 10 5.493 7.840 222.7 0.064Similar to 60S acidic ribosomal protein P2 55635097 10 5.661 7.157 31.3 0.094F-box only protein 2 15812198 10 7.497 4.962 �343.0 0.030Complement factor B 14124934 10 4.749 6.756 101.6 0.065Ig � light-chain VLJ region 21669507 9 7.356 8.073 5.2 0.073Coagulation factor II 30802115 9 4.000 6.313 205.8 0.030Ig �-1 heavy-chain constant region 184749 9 4.000 6.540 347.1 0.030Aldehyde reductase 30582845 8 6.645 4.444 �158.8 0.041Hemopexin 11321561 8 4.442 7.154 515.0 0.030Osteomodulin 37182862 7 6.728 8.053 21.1 0.087Histidine-rich glycoprotein 4504489 7 4.249 6.442 156.0 0.047Transmembrane protein 4 37183321 7 5.280 6.881 39.9 0.093Complement 9 2258128 7 4.000 6.388 244.5 0.030Glutathione S-transferase M2 4504175 6 6.317 4.380 �86.5 0.039D-dopachrome tautomerase 30582779 6 6.699 4.955 �55.4 0.030Inositol(myo)-1 (or 4)-monophosphatase 1 5031789 6 6.023 4.000 �105.4 0.030H1 histone family, member X 5174449 6 6.868 4.364 �319.2 0.003Apolipoprotein A-II 30582411 6 4.494 6.251 57.2 0.047Unknown (protein for IMAGE:4901992) 30353943 6 6.921 4.957 �92.2 0.094Acrosomal serine protease inhibitor 13195769 6 4.000 6.151 141.7 0.030TGF�1-binding protein 107945 6 4.000 6.453 283.7 0.030HINT1 10439439 5 6.475 5.083 �24.6 0.094Osteonectin 4507171 5 4.000 5.551 35.6 0.094Proteasome �1 subunit 12653473 5 6.303 4.257 �111.4 0.030MRCL3 protein 16741043 5 5.318 6.923 40.3 0.091�1 acid glycoprotein 1 1197209 4 4.000 5.857 71.9 0.073Cystatin C 30582517 4 4.000 5.581 38.1 0.082Acid phosphatase 1 isoform c 4757714 4 4.357 5.948 38.9 0.090Similar to fatty acid–binding protein, adipocyte 55630858 4 4.000 5.738 54.7 0.081Small inducible cytokine B10 15012099 3 5.571 4.267 �20.1 0.087HPRT1 47115227 3 5.331 4.000 �21.4 0.073Tenomodulin 11545883 3 5.392 4.000 �24.7 0.073Branched-chain aminotransferase 1, cytosolic 38176287 3 5.820 4.351 �29.5 0.093Transforming growth factor �1 10863873 3 5.210 4.000 �16.2 0.073

* These proteins were selected from the 814 proteins identified from the 14 samples. The following criteria were applied: the protein must beidentified with �3 unique peptides; the protein must be identified in �4 samples in at least 1 group; the difference of mean total ion currents inthe 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).

DIFFERENTIAL PROTEOMIC PROFILING OF HUMAN NORMAL AND OA CARTILAGE 3679

cartilage intermediate-layer protein, and decorin wereamong the most abundant, as measured by the sums ofTIC values of their corresponding peptides. The proteinlist also contains several proteases and their inhibitors,as well as cytokines, kinases, and other signaling mole-cules that are usually present in very low amounts incartilage.

Selection of differentially expressed proteins. Toevaluate the relative changes in the levels of proteinexpression, a variety of criteria were applied, as follows:1) the protein must be identified from �3 uniquepeptides; 2) the protein must be identified in �4 samplesin at least 1 group; 3) the difference in the mean TICvalues in 2 groups must be �5-fold (if the protein wasnot detected in a sample, a TIC value of 104, approxi-mately equivalent to the noise level of the ion trapinstrument, was arbitrarily assigned); and 4) FDR-adjusted (33) P values of �0.10 by t-test (correspondingto raw P values less than 0.03) were applied to furthernarrow down the list of differentially expressed proteins.Among the 814 unique proteins, 595 were identifiedfrom �3 peptides, and 420 of these 595 proteins wereidentified in �4 samples from at least 1 group (seeSupplementary Table 3, available online at http://bioinfo.public.wyeth.com/WHB). One hundred eighty-three of these proteins exhibited a mean TIC change of�5-fold in either direction. However, only 59 met thecriteria after applying the FDR-adjusted P value cutoff(Table 1). The changes in these 59 proteins betweennormal and OA samples are also shown in the cluster-gram in Supplementary Figure 1 (online at http://bioinfo.public.wyeth.com/WHB). For example, HtrAserine protease 11 (HtrA1; also called PRSS11), themost abundant protease in cartilage, was identified in allnormal and OA samples from a total of 29 uniquepeptides (Table 1). Its expression in OA cartilage was�8-fold higher than that in normal cartilage (meanTIC1.8E � 09 versus 2.3E � 08).

The differentially expressed proteins shown inTable 1 represented a wide range of biologic categories,including major serum proteins such as apolipoproteins,hemopexin, fibrinogen, complement components; deg-radative proteases and protease inhibitors such asHtrA1, MMP/tissue inhibitor of metalloproteinases(TIMP), acrosomal serine protease inhibitor, and cysta-tin C; cartilage anabolic effectors such as different formsof transforming growth factor � (TGF�) and TGF�binding proteins; extracellular matrix proteins such asthrombospondin 1; as well as several interesting proteinswhose properties and functions in OA pathophysiologyhave not been well characterized, such as fibulin 3,

osteonectin, secreted modular calcium-binding protein2, vitrin, and tenomodulin.

Bioinformatics characterization of cartilage pro-teins. The proteins identified in cartilage tissue wereanalyzed by a variety of bioinformatics tools. Based onthe TIC values of the 420 proteins (see SupplementaryTable 3) identified in each experimental sample, the 14cartilage samples could be separated into 2 distinctgroups of OA and normal samples (Figure 3). This resultdemonstrated that OA cartilage samples have distinctprotein expression profiles that can be distinguishedfrom the protein profile of normal cartilage. Pathwayanalysis using tools provided by Ingenuity (www.ingenu-ity.com) showed that the 59 differentially expressedproteins in OA (those with an FDR-adjusted P value ofless than or equal to 0.10) cover a broad range of proteinfunctional classes (Figure 4). A significant portion ofproteins 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 of59), and immune response (8 of 59).

Figure 3. Sample correlation using the total ion current values from420 selected proteins, each of which was detected from �3 uniquepeptides in at least 4 samples/group. The color scale ranges from 0 (nocorrelation) to 1 (self–self correlation). OA � osteoarthritis; N �normal.

3680 WU ET AL

DISCUSSION

In this study, we performed differential proteom-ics for the analysis of proteins in human articularcartilage, to elucidate changes in protein expression inpatients with OA. The unique features of our study areas follows. First, this is, to our knowledge, the firstapplication of comparative proteomics to cartilage tissue(as opposed to cultured chondrocytes) from normalindividuals as well as patients with OA. Second, a totalof 814 distinct proteins were identified, and a largeproportion of these proteins and their expressionchanges was identified for the first time from cartilagetissue, thus providing a comprehensive cartilage pro-teome database that is essential in understanding themolecular mechanisms underlying OA. Third, 59 pro-teins displayed significant differences in abundance inassociation with OA. The functions of the proteins withsignificantly different abundance in OA versus normalarticular cartilage may be associated with the pathogen-esis of OA, such as proteolysis, lipid metabolism, im-mune response, cell death, and signal transduction.

A major obstacle in applying proteomics to car-tilage tissue is the heterogeneous nature of cartilageproteins. Articular cartilage contains mainly ECM pro-teins, of which type II collagen and aggrecan comprise

�90% of the total dry mass, whereas chondrocytes, theonly cell type in cartilage, account for �5% of the totalcartilage volume. As a result, the separation ofcrosslinked collagen and highly sulfated proteoglycanaggrecan was critical to the overall success of thestrategy. In fact, neither collagens nor aggrecan waspresent as predominant cartilage proteins in our pro-teomic analyses.

Proteomic analysis of human articular cartilagedemonstrated variability in the abundance of manyproteins between different individuals, as indicated byhighly variable TIC values for an individual protein indifferent samples. The criteria for selecting differentialproteins included not only the confidence of the proteinidentification and the relative TIC changes but also theoccurrence frequencies of the proteins within a group.These stringent criteria ensured that a selected proteinwas truly present in a majority of samples within a group,and that the altered abundance was not random. Tofacilitate the data processing, undetected (“absent”)proteins were arbitrarily assigned a TIC value of 104,which was approximately equivalent to the instrumentnoise level and significantly lower than most of the TICvalues observed in the data set. Protein expressionchanges from normal to OA groups were assessed by the

Figure 4. Ingenuity pathway analysis results, showing functional categories of the 59 differentially expressed proteins in osteoarthritis (OA) versusnormal articular cartilage. The proteins are displayed according to physiologic function. The solid and open bars represent proteins that areincreased 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.

DIFFERENTIAL PROTEOMIC PROFILING OF HUMAN NORMAL AND OA CARTILAGE 3681

logarithmic difference of their mean TIC values, and thesignificance of such changes was assessed. The P valuefor the mean TIC change for each protein was calcu-lated, and only those with an FDR-adjusted P value lessthan or equal to 0.1 were selected, to ensure that thechanges in expression for individual proteins betweenthe normal and OA groups were truly significant. Thisresulted in 59 proteins that were significantly changed(Table 1).

The etiology and pathophysiology of OA diseaseare complex and only partially understood. However, itis generally accepted that an imbalance between ana-bolic and catabolic pathways in matrix turnover contrib-utes significantly to the mechanism (1,3,4,6). The in-creased synthesis of degradative enzymes such as MMPand ADAMTS may largely account for the unbalancedbreakdown of the cartilage ECM. Several cartilage pro-teases and protease inhibitors were identified in thecurrent study. Among these, the levels of HtrA1,TIMP-2, MMP-2, cystatin C, and acrosomal serine pro-tease inhibitor were all significantly elevated in OAcartilage, while the levels of many others were alsoincreased, but to a lesser extent (Table 1 and Supple-mentary Table 3). HtrA1 was the most abundant pro-tease identified in cartilage tissue, as estimated by theTIC value generated by unique peptides. This resultconfirmed previous findings reported by Hu et al (35)and Grau et al (36), showing elevated expression ofHtrA1 at the transcriptional level in OA articular carti-lage, and elevated levels of HtrA1 protein (�7-foldabove normal) in synovial fluids obtained from patientswith OA.

In vitro studies, including those by our group,have shown that HtrA1 is able to digest several cartilagematrix proteins, including aggrecan, decorin, fibromodu-lin, and soluble type II collagen (37), and to inhibitTGF�-induced matrix synthesis by chondrocytes (YangZ, et al: unpublished observations). Chondrocytes alsosynthesize a variety of MMPs, including collagenases(MMP-1, MMP-8, and MMP-13), gelatinases (MMP-2and MMP-9), stromelysin 1 (MMP-3), and membrane-type MMPs. MMP-1, MMP-2, and MMP-3 were identi-fied in this study, but only MMP-2 was altered inquantity in OA cartilage. Cysteine proteinases (such ascathepsins B, H, K, L, and S) represent another impor-tant enzyme family that might play a role in cartilagematrix destruction. Several cathepsins, particularly ca-thepsin K, have been shown to participate in OA carti-lage degradation (38–40). Cystatin C, a potent extracel-lular inhibitor of cysteine proteinases, has also beenlinked to arthritis. Results from transcriptional profiling

of cysteine proteinases and their inhibitors in a trans-genic mouse model of OA showed that up-regulation ofcathepsin K, but not cystatin expression, coincided withthe onset of articular cartilage damage (41). However,our data strongly indicated that the level of cystatin C isincreased in human OA cartilage.

The levels of several plasma proteins such asapolipoproteins (Apo A-I, II, IV, and Apo H) aresignificantly elevated in OA cartilage. The functionalrole of lipoproteins and their receptors in OA and otherjoint diseases has been described in the literature(42,43). The observed up-regulation of these proteins inOA may also imply their involvement in disease mech-anism that has not yet been fully investigated.

Finally, another of the interesting protein fami-lies identified in cartilage tissue comprised the fibulins.Fibulins are ECM proteins (44), and 6 different familymembers have been reported. All are characterized by aunique C-terminal fibulin-type module and several re-peated epidermal growth factor–like domains. We iden-tified 3 different fibulin proteins in cartilage: fibulin 1D,fibulin 2, and fibulin 3 (Figure 5). Fibulin 2 was identi-fied in only 2 OA samples and 1 normal sample, at verylow levels. In contrast, fibulin 1D and fibulin 3 wereidentified with strong abundance and were primarilyobserved in gel bands consistent with their intact molec-ular masses. The expression of fibulin 1D was slightlyelevated in OA cartilage (mean TIC3.1E � 08 in OAcartilage versus 1.2E � 08 in normal cartilage), whereasexpression of fibulin 3 was strongly increased in OAcartilage compared with normal cartilage (meanTIC3.6E � 08 versus 3.9E � 06).

Fibulin 1D binds to proteoglycans and was re-cently reported as being a cofactor of ADAMTS-1, aninhibitor of angiogenesis and a mediator of proteolyticcleavage of aggrecan. When colocalized in vivo with

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

3682 WU ET AL

ADAMTS-1, fibulin 1D was shown to enhance thecapacity of ADAMTS-1 to cleave aggrecan (45). Arecent study demonstrated that fibulin 3 is a bindingpartner of TIMP-3, which is a known inhibitor of MMPsand aggrecanases (46). Collectively, such evidence indi-cates that the fibulin family might play an important rolein cartilage metabolism.

To our knowledge, the current study is the firstcomprehensive proteomic analysis of soluble proteinsextracted from normal and OA cartilage. It provides themost complete catalog of the proteins involved in cellu-lar metabolism and organization, as well as proteinsparticipating in the regulation of ECM synthesis andturnover of articular cartilage. Although the presentfindings are based on a limited number of study subjects,the results, from the protein level, clearly reflect thecomplex pathophysiology of OA. This study providesnew insights for identifying novel OA mediators that canbe potential therapeutic targets or biomarkers for OA.

ACKNOWLEDGMENTS

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

AUTHOR CONTRIBUTIONS

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

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