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Originally published In Press as doi:10.1074/mcp.T600007-MCP200 on May 9, 2006.
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Molecular & Cellular Proteomics 5:1984-1995, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Methodologies

Establishment of a Reliable Method for Direct Proteome Characterization of Human Articular Cartilage*

Jean-Baptiste Vincourt{ddagger},§, Frédéric Lionneton{ddagger}, Gueorgui Kratassiouk, François Guillemin, Patrick Netter{ddagger}, Didier Mainard{ddagger} and Jacques Magdalou{ddagger}

From the {ddagger} Laboratoire de Physiopathologie et Pharmacologie Articulaires, Faculté de Médecine, Unité Mixte de Recherche (UMR) 7561 CNRS-Université Henry Poincaré (UHP), 9 Avenue de la Forêt de Haye, BP 184, 54505 Vandoeuvre-lès-Nancy Cedex, France and Centre Alexis Vautrin, Centre de Recherche en Automatisme de Nancy UMR 7039, CNRS-Institut National Polytechnique Lorrain-UHP, 54500 Vandoeuvre-lès-Nancy, France


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma.

Articular cartilage is a unique, hypocellular, and avascular tissue, mostly made of extracellular collagens and proteoglycans (PGs),1 whose physical properties support frictionless movements and load-bearing capacity of the articulation (1). Homeostasis of this dense, complex extracellular matrix requires perpetual and regulated synthesis/degradation of collagens and PGs by a single cell type, chondrocyte, which accounts for only 1% of the volume in human cartilage (2).

Degeneration of cartilage accompanied with partial loss of articular function is very common worldwide. The cost of all arthritic diseases together was estimated to be more than 100 billion dollars to the United States society for the single year of 1997 (3) and is currently increasing due to the aging of human populations in developed countries. In particular, osteoarthritis, which is characterized by long term breakdown of the cartilage with no intrinsic inflammatory origin, affects the majority of people over 65 years (4). Yet little medication for its treatment is available. Analgesic and anti-inflammatory drugs are used for symptomatic treatment of the associated pain and inflammation without any effect on the progression of the disease (4). Therefore it is necessary to better understand, from a molecular point of view, the early events that lead to osteoarthritis and to identify target proteins, which should provide a rationale for the design of new drugs.

Another challenge in the field of cartilage biology is the diagnosis and treatment of malign chondrogenic tumors (5, 6). Chondrogenic neoplasms are a group of tumors that form cartilage because their cells share much of a chondrocyte phenotype. "Chondrosarcoma" is a general term used to describe, among chondrogenic neoplasms, those that exhibit malignancy. Their early diagnosis after histological analysis is very difficult due to their similarity to benign tumors, which are much more frequent (5). Distinction between the two is important, however, because chondrosarcoma, unlike benign chondroma, should be treated surgically. It is thus critical to identify unequivocal molecular markers of chondrosarcoma (5, 6).

In these respects, most efficient strategies aiming at identifying molecular markers and/or pharmacological targets of cartilage pathologies were based on transcriptional analyses (7–9). These, however, do not directly predict variations of protein levels or post-translational modifications. Therefore, direct, 2-DE-based differential proteomics of whole, healthy, and pathological tissues is an attractive alternative (10, 11). In the case of cartilage, however, such an approach is difficult to perform (12) due to its high content of PGs and collagens (13). These macromolecules impair isoelectric focusing and mask minor, cellular proteins. Consequently proteomic analysis of cartilage has so far been performed under either of two restrictive conditions. The one consists of cartilage enzymatic digestion followed by monolayer primary culture of chondrocytes in fetal calf serum (14), which are likely to introduce artifactual gain or loss of differentiation markers (15–17) and do not clarify changes in the extracellular matrix. The other is based on analysis of proteins secreted by cartilage explants into culture medium (13). It is technically complex, requires much starting biological material, and allows detection of only a minority of cartilage constituents.

To provide a more representative map of the cartilage proteome, we developed a method allowing selective extraction of proteins directly from human articular cartilage and their subsequent resolution by 2-DE. This novel strategy allowed us to generate sufficient amounts of protein extracts from 500 mg or less of macroscopically normal cartilage and to resolve at least 600 spots on 2D gels after silver staining. Extraction was performed efficiently and reproducibly from various sources of pathological tissues from several joints. Interestingly proteins were extracted more efficiently when the method was applied to a chondrosarcoma sample presumably due to the higher cellularity of this tissue. MS analysis of 191 spots revealed a broad diversity in the protein identity and function. This protocol provides a powerful alternative to previous strategies aiming at characterizing cartilage phenotypes at a molecular level. It will be especially helpful at achieving differential analysis of cartilage in physiopathological conditions for identification of protein states that are altered as a cause or a consequence of the diseases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Procurement and Processing—
This study was performed in conformity with the declaration of Helsinki principles. Articular cartilage was obtained from Service d’Orthopédie Chirurgicale (Centre Hospitalier Universitaire, Nancy, France) in the context of amputation and prosthesis replacement. The chondrosarcoma sample was obtained from the same source in the context of tumor amputation. The tumor was diagnosed and graded by the Service d’Anatomie et Cytologie Pathologique from Centre Hospitalier Universitaire, based on radiological and histological analysis. Written informed consent was given by the patient. Cartilage samples were kept in cold saline for transportation and immediately processed. Macroscopically normal cartilage of various pathological sources (Table I) was dissected and washed extensively in cold Dulbecco’s PBS with eventual curettage for removal of all blood cells, wiped on paper towels, and frozen at –80 °C until further use. For chondrosarcoma, regions with macroscopically visible vasculature were removed to avoid contamination by endothelial and blood cells.


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  		TABLE I Cartilage samples used in this study

For each sample, gender (F, female; M, male), age, and pathology of the patient are given. Also extraction yield of the method, as estimated by the acidified Bradford assay, is annotated.

 
Protein Extraction and Preparation—
Cartilage was soaked with cartilage extraction buffer (500 mM NaCl, 50 mM HEPES, pH 7.2, Complete protease inhibitor (Roche Applied Science)) and frozen on a cryotome plate (HM440E, Microm) at –30 °C. The tissue was cut into 10-µm slices and suspended in cold extraction buffer at 100 mg of cartilage/ml. Samples were agitated at room temperature for 1 h. Extracted material was clarified upon centrifugation (6,000 x g for 5 min), and 1% (w/v) CPC was added. PGs and CPC were allowed to aggregate (1 h at room temperature under agitation) and removed by centrifugation (6,000 x g for 5 min). The resulting supernatant was submitted to chloroform/methanol precipitation (18) with a slight modification. After the first centrifugation (14,000 x g for 30 min), both liquid phases were removed. The insoluble material was resuspended in 4 volumes of methanol and centrifuged again in identical conditions. Pellets were resuspended in 1 volume of methanol, transferred to safe lock Eppendorf tubes, spun down, dried, and resuspended in 200 µl of 2D Sample Solution (2DSS; 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.2% (w/v) Triton X-100, Complete). For protein concentration measurement, samples diluted in a final volume of 10 µl of 2DSS were acidified with 2 µl of 0.1 N HCl and processed for Bradford assay (19) using bovine serum albumin solubilized in 2DSS as a standard. Protein samples were diluted to 1 µg/µl in 2DSS supplemented with 1% (w/v) DTT and trace bromphenol blue, aliquoted at 200 µg/sample, and kept frozen at –80 °C until further use.

Two-dimensional Electrophoresis—
Cartilage protein samples (200 µg) were thawed and agitated for 2 h, ultracentrifuged (100,000 x g for 30 min at room temperature) for insoluble material removal. The supernatant was allowed to rehydrate IPG strips (Bio-Rad, 24 cm, pH 3–10, 10 h at room temperature) in a final buffer volume of 450 µl (2DSS supplemented with DTT and bromphenol blue). Electrofocalization was performed in a Protean IEF cell (Bio-Rad) through a single step program according to the manufacturer’s recommendations. Strips were cut on each side for obtaining strips of pH range 4.5–8. Disulfide bonds of proteins were reduced by 10-min incubation in 4 ml of equilibration buffer (6 M urea, 2% SDS, 50 mM Tris, pH 8.8, 20% (v/v) glycerol) containing 130 mM DTT. Free sulfhydryl groups were alkylated by treatment with 135 mM iodoacetamide for 10 min in equilibration buffer. Strips were transferred to the top of 12% (w/v) acrylamide denaturing gels poured in Criterion cassettes (Bio-Rad) and run at 17 mA.

Gel Staining—
Gels were fixed for 2 h in two successive baths of 30% (v/v) methanol, 10% (v/v) acetic acid. For silver staining, gels were extensively washed in water overnight. Gels were sensitized for 2 min in 0.02% (w/v) sodium thiosulfate, quickly washed twice in water, and impregnated in 0.2% (w/v) silver nitrate for 1 h. Excess silver nitrate was washed quickly, and staining was developed with 3% (w/v) potassium carbonate, 2.5 mg/liter sodium thiosulfate, 0.025% (v/v) formalin and stopped with 1% (w/v) acetic acid for 30 min. Gels were washed in water and kept in water at 4 °C. For colloidal Coomassie Brilliant Blue staining, gels were washed several times in fixative and incubated in staining solution (8% (w/v) ammonium sulfate, 1.6% (w/v) phosphoric acid, 0.08% (w/v) Coomassie G250) overnight. The next day, gels were washed extensively in water until optimal contrast was obtained.

Reproducibility Assay—
Extraction of proteins was performed three times independently in three successive experiments, each from 500 mg of the same cartilage sample. Protein concentration of each extract was quantified, and 200 µg were used for 2-DE. Gels were stained with silver nitrate and scanned. Qualitative and quantitative comparison of triplicates was performed using the PDQuest software (Bio-Rad) on a relatively central zone of the gels to avoid board effects and poor resolution in the pI dimension. For qualitative assay, the software was used in the automatic mode with subsequent manual correction. For semiquantitative comparison, integrated optical densities of automatically detected, qualitatively conserved spots were normalized to the total density of validated spots from the corresponding gel. The normalized coefficient of variation (CV) of each spot was calculated, and the percentage of spots that fall within a defined CV cutoff value (ranging from 0.3 to 1) is reported in Table II.


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  		TABLE II Qualitative and quantitative assessment of the reproducibility

Extraction was performed in separate triplicates from samples S9 and S6 (see Table I). Resulting gels were scanned and analyzed using PDQuest software as mentioned under "Experimental Procedures." Analysis was restricted to a relatively central area of the gels to avoid board effects and poor resolution in the pI dimension. Spots were detected automatically and corrected manually. They were counted and matched between gels. The number of detected spots is indicated as mean ± S.D. Unmatched spots are expressed as their average percentage relative to total spot numbers ± S.D. For quantification, spots common to all triplicates within each group were quantified and normalized to the total density of validated spots from the corresponding gel. For each spot, the CV between all three gels was calculated. The proportion of spots with a CV lower than the indicated cutoff is shown in the corresponding column as a percentage.

 
Trypsic Digestion—
After excision, spots (2-mm diameter) were processed. All washes were performed in 30 µl for 15 min. Spots were washed twice in water and twice in fresh 50 mM ammonium bicarbonate, 50% (v/v) acetonitrile. Spots were dehydrated for 1 min in 100% (v/v) acetonitrile, washed once again in 50 mM ammonium bicarbonate, 50% (v/v) acetonitrile, and dried in a SpeedVac (RC10.10, Jouan) for 15 min. Each sample was digested overnight at 37 °C in 7 µl of sequencing grade modified trypsin (Promega) solution (12.5 ng of trypsin/µl in 25 mM ammonium bicarbonate).

Mass Spectrometry—
After digestion, the trypsin solution containing peptides was collected. For further peptide extraction, 8 µl of 50% (v/v) ACN, 2% (v/v) TFA were added onto each spot and sonicated for 10 min in a waterbath sonicator (Transsonic 570, Elma). ACN (100%, v/v) was added (2 µl). Both solutions containing peptides were pooled together. Samples (0.6 µl) were laid on a MALDI target (AnchorChip, Bruker Daltonik) using the dried droplet method with {alpha}-cyano-4-hydroxycinnamic acid as a matrix. Peptides were analyzed by MALDI-TOF mass spectrometry using an Ultraflex mass spectrometer (Bruker Daltonik). Measurements were externally calibrated with different peptide standards. When required, spectra were internally calibrated with peptides resulting from trypsin autolysis. Monoisotopic peptide masses were assigned, and searches were performed using the MASCOT software (Matrix Science, Oxford, UK) with carboxamidomethylation of cysteines as fixed modifications and methionine oxidations as variable modifications searching the Mass Spectrometry Protein Sequence Database (MSDB) and Swiss-Prot (www.expasy.ch/sprot/) databases with a mass tolerance of 50 ppm.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of a Method for Proteome Extraction and Analysis from Human Arthritic Articular Cartilage—
Intrinsic limitations of classical proteomic approaches for studying cartilage are mostly due to the high content in this tissue of PGs and collagen species, which strongly affect isoelectric focusing (13) and impair detection of minor proteins. PGs and collagens, however, have particular biochemical properties, based on which we could design a method allowing their exclusion in an exhaustive protein extraction system.

First because cartilage is solid, protein extraction requires disassembling of the tissue to allow its impregnation by the chosen buffer. This is classically done by total dissociation of the tissue (20). On the contrary, we preferred to preserve the initial structure of the matrix as much as possible to minimize mechanical extraction of collagens and PGs. For this purpose, cartilage was dissected through cryotome section into 10-µm slices.

Collagen molecules assemble into large, macroscopic, insoluble fibers depending on multiple, well organized hydrogen bonds (21). As such, their cohesion is strongly affected by chaotropic agents, such as urea, which is classically used for protein solubilization prior to 2-DE analysis (Fig. 1A, right lane). In contrast, buffers containing high NaCl concentration are relatively permissive for collagen fibers cohesion while allowing "solubilization" of most other protein components of cartilage (Fig. 1A, left lane). The obtained extract was not resolvable by 2-DE presumably due to its high remaining PG content (Fig. 1B). We took advantage of the previously described method for removal of PGs by selective precipitation with CPC (13), which we found to be efficient at NaCl concentrations not exceeding 500 mM. Finally because CPC is an ionic detergent, it should be removed from the extract as well as salts as their presence would impair isoelectric focusing. For this purpose, we purified the resulting protein extract by methanol/chloroform precipitation (18) at room temperature, which also removes nucleic acids and lipids. The resulting pellet was dried and solubilized in a classical 2-DE loading buffer, providing a protein extract that was efficiently resolved by 2-DE, as shown on Fig. 1C. A standardized method for cartilage proteomic analysis, described in detail under "Experimental Procedures," was designed based on these findings.


Figure 1
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  		FIG. 1. Principles of cartilage proteome extraction method. A, comparison of extraction buffers. Human knee arthritic cartilage (100 mg) was washed in saline and subjected to cryotome section and resuspension for 1 h in 500 µl of extraction buffer containing 50 mM HEPES, pH 7.2, and either 500 mM NaCl or 2 M urea, 4% (w/v) CHAPS. After removal of insoluble material by centrifugation at 6,000 x g for 5 min, 125 µl of 5x Laemmli sample buffer was added. Samples were boiled, and 50 µl were separated on a 10% (w/v) acrylamide denaturing gel subsequently stained with Coomassie Brilliant Blue. Arrows indicate expected molecular masses of mature collagen II and aggrecan. B and C, cartilage proteins were extracted in 500 mM NaCl-containing buffer as described in A. In B the protein content was directly precipitated with methanol/chloroform, whereas in C PGs were first aggregated with 1% (w/v) CPC according to the "Experimental Procedures," and the protein content was subsequently precipitated with methanol/chloroform. Extracts were then solubilized in 2DSS and assayed for protein concentration, and 200 µg were processed for 2-DE. Silver staining was performed.

 
Efficient Proteome Extraction from Cartilage of Various Origins and Pathological States—
Because such a method has many potential uses in studying cartilage biology, we assayed its capability on various kinds of human cartilaginous tissues. As shown in Table I, these include articular cartilage from hip, knee, and talus affected by various diseases. Also a sample of low grade chondrosarcoma was included. Cartilaginous tumors of low grade possess the same intrinsic incompatibilities with classical proteomic approaches as articular cartilage because they synthesize a comparable extracellular matrix (5, 22). Consequently biochemical methods that apply to articular cartilage should also apply to such tumors. All samples were processed identically and provided extracts that could be well separated by 2-DE (Fig. 2). Amounts of protein extract obtained from all articular cartilage samples were very similar (see Table I) with an average extraction yield higher than 400 µg of extract/g of cartilage. Also the extraction efficiency was found to be reproducible (S.D. ≤ 10%) when performed twice independently on the same cartilage sample (data not shown). When applied to a chondrosarcoma sample, the method was dramatically more efficient as its extraction yield reached 2.4 mg of proteins/g of tissue. Because the major macroscopically visible difference between cartilaginous tumor and articular cartilage is a higher cellularity of chondrosarcoma (22), it is likely that (i) the extraction yield increases with cellularity and (ii) intracellular proteins occupy a high proportion of the extract.


Figure 2
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  		FIG. 2. Electrophoretic profiles of cartilage proteomes from various tissues. Protein extracts (200 µg) from various tissues (see Table I) were loaded on 2D gels. 2-DE analysis was performed as indicated under "Experimental Procedures." A, S3; B, S9; C, S2; D, S4; E, S8; F, S6.

 
Reproducible Extraction of Proteins from Cartilage Samples—
Extracts obtained with this method exhibited a relatively similar electrophoretic pattern, in the sense that most spots appeared identical from one sample to another, according to their respective shapes and coordinates on 2D gels (Fig. 2). Reproducibility is a recurrent issue when using 2-DE because 2-DE itself introduces substantial variation in the number, intensity, and localization of spots on 2D gels (23, 24). To challenge the reproducibility of the proposed method, two of the samples shown in Fig. 2 were subjected to independent, triplicate extractions, 2-DE, silver staining, and subsequent comparative analysis. We used an osteoarthritic cartilage sample and the chondrosarcoma sample (Fig. 2, B and F). For each tissue, reproducibility of the results was assessed qualitatively and quantitatively (Table II). The spot counts were 558 ± 39 for sample S9 and 613 ± 22 for sample S6, showing little variation in this respect. Spots that could not be matched from one gel to another (from the same sample) were counted and found to represent 18%, on average (Table II), of all spots counted from the corresponding gels. This value was close to those reported for the reproducibility of 2-DE using silver staining detection (25) and other comparable methods (23), showing good qualitative repeatability of the method. To evaluate the quantitative reproducibility, the content of each conserved spot was estimated as described under "Experimental Procedures." For each spot, the CV of the quantity, as measured between all three gels, was calculated. Spots were classified as populations that have a CV falling within a defined cutoff value (ranging from 0.3 to 1, Table II) as suggested previously (23). According to Reed et al. (26), the repartition of CV values found in our study allows searching for 4-fold expression differences between two samples with as little as 1.6% false positive spots. Therefore, using populations of samples, the proposed procedure is suitable for the identification of protein markers of cartilage diseases.

Toward Exhaustive Proteomic Analysis of Articular Cartilage—
One important criterion for defining the ability of the method to enlighten cartilage phenotypes is the variety in identity and function of analyzed proteins. As an initial effort to emphasize heterogeneity of the extract, 220 spots were randomly chosen among those of highest staining for mass spectrometry analysis after tryptic digestion. For ease of procedure, this was done from a sample of femoral arthritic cartilage after colloidal Coomassie Brilliant Blue staining (Fig. 3). The content of several spots could not be identified despite detection of normal mass spectra by MS presumably due to abundant glycosylations. The content of 191 spots, however, was unambiguously identified and annotated on Fig. 3. Also identified proteins were classified based on their known physiological functions in Table III. Those identified proteins correspond to 127 distinct gene products. The ratio between the two (127:191) denotes respectable variety of proteins in the extracts. Eighty-two of these have intracellular roles, ranging from general cellular physiology to signal transduction, stress response, and matrix processing. We also found 16 proteins from the extracellular matrix, mostly in an unprocessed, intracellular form. Seven complement factors were identified, including both premature and processed forms, in agreement with previous demonstration of their expression by chondrocytes in vivo (27). In total, 105 identified proteins actually originated from inside the chondrocytes, confirming a selective extraction of intracellular proteins. Finally 22 proteins are commonly considered as plasma components, yet synovial fluids were previously shown to contain those proteins as well (28). Because synovial fluids do communicate directly with cartilage, unlike plasma, we classified them as "synovial fluid" components.


Figure 3
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  		FIG. 3. 2D map of the human articular cartilage proteome. Protein extract (450 µg) from sample S3 was separated by 2-DE as described under "Experimental Procedures" and stained with colloidal Coomassie Brilliant Blue. The content of 191 spots was identified by MS after tryptic digestion. Spots are indicated by arrows, and their names are abbreviated. Detailed analysis is compiled in Table III.

 

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  		TABLE III Functional classification of proteins identified by MS

Classification was performed according to the Swiss-Prot database. Abbreviations used for annotation of the proteomic map in Fig. 3 are listed. The General Identifier (GI) from the National Center for Biotechnology Information (NCBI) and full name of the corresponding protein are indicated as well as its theoretical molecular weight/pI. Also the number of peptide masses isolated from the corresponding spot that match the reference sequence and their coverage of the sequence are annotated. ER, endoplasmic reticulum; EGF, epidermal growth factor; HBsAg, hepatitis B surface antigen; TGFß, transforming growth factor ß; HS, heparan sulfate.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Despite an increasing need for large scale characterization of the cartilage proteome (29), classical and non-classical proteomic approaches for cartilage have been impractical until recently (12) due to the particular content of this highly specialized tissue (13). Indirect approaches have been used consisting of the analysis of proteins secreted by cartilage when incubated in culture medium (13) or proteins extracted from chondrocytes in culture (14).

Here we describe a novel method allowing direct 2D gel-based proteomic characterization of cartilaginous tissues. This method was found to be qualitatively and quantitatively reproducible (Tables I and II) using articular cartilage of several joints affected by some of the most common cartilage-related pathologies, and extracted content could be well separated by 2-DE (Fig. 2). The method was also found to be pertinent to the analysis of cartilaginous tumors. Electrophoretic profiles of more than 600 protein spots were all globally similar, providing a "cartilaginous tissue signature" in the sense that major spots were identically present on stained gels made from any sample.

A reference sample was analyzed by MS, revealing the identity of 191 of its most intensely stained spots seen on gel. The extracted proteins had very broad spectrum of physiological functions, most being intracellular. Indeed the electrophoretic pattern of extracts described here shared much homology with that of the previously described proteome of cultured chondrocytes (14). This similarity appears strikingly when comparing lists of identified proteins: 41 of 93 proteins identified by Ruiz-Romero et al. (14) were also found in our study (Table III). Our approach, however, presents several major advantages. From a practical point of view, our method allows the use of frozen, eventually non-sterile cartilage samples. This is obviously impossible for alternatives using cell culture; also it is faster and requires no specific material. More importantly, our method provides direct analysis of the in vivo status of chondrocytes. Cultivation of chondrocytes as monolayer in fetal calf serum-containing media is a very important tool for studying molecular mechanisms of their physiology. When possible, however, its use should be avoided in the context of detailed expression studies as it introduces both variability and loss of differentiation markers (15–17). In this sense, our protocol constitutes much of a conceptual improvement compared with those that involve chondrocyte culture. One possibly direct consequence of this improvement is the finding of several complement factors originating from chondrocytes (27). These are not only obvious regulators of the immune system but also apparent modulators of cartilage matrix degradation. As such, they have potential involvement in cartilage degenerative diseases and are interesting to be taken under consideration (summarized and discussed in Refs. 27, 30, and 31)).

There is one single precedent of a method allowing direct proteomic analysis of cartilage (13) based on "secretion" of de novo synthesized proteins from cartilage explants into culture medium. This work first described the principle of PG removal by CPC precipitation. However, because this study was focused on diffusing proteins, cellular components were poorly represented; only 27 different proteins were analyzed, most of which corresponded to cartilage matrix components and regulatory molecules. Indeed the authors argued that proteins originating from synovial fluids should not be taken into account because they are not produced by chondrocytes. On the contrary, we considered them as taking part of the general and pathological physiology of cartilage and, as such, potentially informative. Additionally intracellular proteins are much better represented with our method because it was designed for their targeted extraction. As a result, our approach provides a broad and detailed look at molecular events that take place in cartilage.

The most obvious limitation of our protocol is the large amount of albumin remaining in purified extracts (Fig. 3, estimated to 30%). This results in masking of other proteins of similar molecular weight/pI and reduces the quantity of informative proteins to be loaded on isoelectric focusing strips. For some applications, users might want to include an additional step of immunoaffinity-based removal of albumin as is classically done for plasma samples (32). However, we found it difficult to remove more than 80% of the albumin content. Also removal of albumin seemed to be a cause of quantitative variability for certain protein spots (unidentified, data not shown). Furthermore albumin was not found at such a high level from all kinds of cartilaginous tissues (see Fig. 2). In particular, albumin depletion might be avoided in the context of cartilaginous tumor characterization, which we are currently conducting without major difficulties in this regard.

In summary, our newly set-up protocol combines advantages of both previously proposed methods for global cartilage proteome analysis together with additional ones. It is achievable from low quantities of starting material, a major improvement, particularly for studying cartilage tissue from small joints and tumors (Fig. 2, E and F). Also it allows convenient practices, such as sample freezing prior to processing. This procedure was found to be reproducible from several sources of articular cartilage differing in joint origin and pathological states. This method will be useful for the development of proteomic strategies aiming at defining key molecular events of cartilage pathologies.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Ouzzine and Dr. L. Grossin, from our laboratory, and S. Shaw, from NCI, National Institutes of Health, Bethesda, MD, for careful reading of the manuscript and constructive comments.


  FOOTNOTES
 
Received, February 14, 2006, and in revised form, April 10, 2006.

Published, MCP Papers in Press, May 9, 2006, DOI 10.1074/mcp.T600007-MCP200

1 The abbreviations used are: PG, proteoglycan; CPC, cetylpyridinium chloride; 2D, two-dimensional; 2-DE, two-dimensional electrophoresis; CV, coefficient of variation. Back

* This work was supported in part by the Association pour la Recherche contre le Cancer (ARC), Ligue Régionale contre le Cancer, Région Lorraine, and Institut Federatif de Recherche 111 Bioingénieurie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by ARC. To whom correspondence should be addressed. Tel.: 33383683950; Fax: 33383683959; E-mail: Jean-Baptiste.Vincourt{at}medecine.uhp-nancy.fr


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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