Strategies to optimize two-dimensional gel electrophoresis analysis of the human joint proteome

Talanta 80 (2010) 1552–1560

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Strategies to optimize two-dimensional gel electrophoresis analysis of the human joint proteome Cristina Ruiz-Romero, Valentina Calamia, Vanessa Carreira, Jesús Mateos, Patricia Fernández, Francisco J. Blanco ∗ Proteomics Unit and Associated Node to ProteoRed, Osteoarticular and Aging Research Lab, INIBIC-Hospital Universitario A Coru˜ na, A Coru˜ na, Spain

a r t i c l e

i n f o

Article history: Available online 22 May 2009 Keywords: Proteomics 2-DE Joint Cartilage Chondrocyte Synovium Synovial fluid

a b s t r a c t Due to the complex structure of the articular joint, it requires great effort to fully understand joint disease pathogenesis. The proteomic analysis of articular joint tissues could contribute greatly to our insight into the endogenous control mechanisms of matrix turnover and the unravelling of the molecular and cellular mechanisms involved in the progression of the arthritides. To date, most proteome analysis strategies use the two-dimensional gel electrophoresis (2-DE) technique to separate proteins according to their isoelectric point, molecular mass, solubility and relative abundance. In this work, we describe optimization of human joint sample preparation techniques to obtain high quality 2-DE maps of human joint tissues (cartilage and synovium), cells (chondrocytes and synoviocytes) and synovial fluid. These techniques improve the performance of gel-based differential proteomic analysis, and facilitate the application of proteomics to rheumatology studies. © 2009 Elsevier B.V. All rights reserved.

1. Introduction A comprehensive understanding of the protein pathways involved in normal and disease states is required if we are to effectively treat disease. Proteome analyses not only aim to identify changes in protein expression, but also protein interactions, posttranslational modifications and protein distribution. Proteomics, therefore, has become a useful technology for gaining an understanding of the complex and unknown processes that participate in joint disease pathogenesis. More efficient strategies are needed to obtain high throughput proteomics analysis of tissues and cells, in order to gain insight into metabolic dysregulations and structural changes that lead to disease development. Many proteomic research strategies currently employ twodimensional gel electrophoresis (2-DE) to separate proteins according to their isoelectric point (pI), molecular mass, solubility and relative abundance [1]. The gels are then stained with Coomasie brilliant blue [2], silver nitrate [3] or fluorescent dyes such as SYPRO Ruby or ruthenium II tris(bathophenanthroline disulfonate) [4,5] to visualize the protein spots. Digitized gel images are used for analysis and specific proteins to be identified are removed from the gel

∗ Corresponding author at: Unidad de Investigación Osteoarticular y del Envejecimiento, Laboratorio de Investigación, INIBIC - Complejo Hospitalario Universitario ˜ C/ Xubias, 84, 15006 A Coruna, ˜ Spain. Tel.: +34 981 178272; A Coruna, fax: +34 981 178273. E-mail address: [email protected] (F.J. Blanco). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.05.022

and digested using a specific protease, usually trypsin. The proteins are subsequently identified using mass spectrometry [6]. The high resolution of this technology makes it suitable for the analysis and parallel quantitative profiling of complex protein mixtures, such as whole cell lysates or tissues. Unlike other proteomic approaches that work with peptides, such as those methods based on liquid chromatography with tandem mass spectrometry (LC–MS/MS), 2DE provides information about molecular weight and isoelectric point, and delivers a map of intact proteins that reflects changes in isoforms, post-translational modifications and protein expression levels. Early limitations of 2-DE have been largely overcome, not only with respect to reproducibility and handling, but also with the resolution of proteins that are hydrophobic or that exhibit extreme pI values. This has been possible mainly with the development of immobilized pH gradients (IPGs) that enable the analysis of very acid or alkaline proteins (IPGs between 2.5 and 12), and also provide increased resolution and allow detection of low abundance proteins using narrow-overlapping IPG strips. Because of this increased resolution, 2-DE can resolve more than 5000 proteins simultaneously using the proper gel size, sample characteristics and pH gradient. 2-DE technology is now able to detect and quantify less than 1 ng of protein per spot. Joint diseases are the leading cause of disability in people over 55 years old. These diseases include degenerative pathologies such as osteoarthritis (OA), autoimmune diseases such as rheumatoid arthritis (RA) or psoriatic arthritis (PA), and metabolic disorders such as gout. While all these pathologies progress with joint inflammation and pain, the patterns differ depending on the type and

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location of the arthritic disease. Considering the complex structure of the articular joint, intensive effort is required to gain an understanding of joint disease pathogenesis. The proteome analysis of articular joints would be very useful to gain insight into the endogenous control mechanisms of matrix turnover in joint tissues, and to unravel molecular and cellular mechanisms that contribute to disease progression. In this paper we describe the sample preparation techniques we have found useful for the study of human joint proteomics using 2-DE for the analysis of joint tissues, cells and fluids. 2. Materials and methods 2.1. Reagents and chemicals Culture media and fetal calf serum (FCS) were from Gibco BRL (Paisley, UK). Culture flasks were obtained from CoStar (Cambridge, MA, USA). 2-DE electrophoresis materials, including IPG buffer and strips were from GE Healthcare (Uppsala, Sweden). Anti-human serum albumin (HSA) and immunoglobulin G (IgG) resin were from Vivascience (Sartorius, Germany). Unless otherwise indicated, all other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Collection of cartilage, synovial membrane, joint cells and synovial fluid Fig. 1 shows protocols followed to obtain joint tissue samples for proteomic analysis. Normal human knee and hip samples were obtained from adult donors, either from joint surgery or autopsy, who had no history of joint disease and macroscopically normal articular tissues. All donors or donor families gave written informed consent for the tissue donation. This study was approved by the Ethics Committee of Galicia (Spain). Synovial membranes were extracted using biopsy forceps, rinsed in sterile phosphate-buffered saline (PBS), weighed, and either frozen at −80 ◦ C or processed for synoviocyte isolation. Cartilage surfaces of femoral condyles were rinsed thoroughly with sterile saline; parallel sections 5 mm apart were then excised using

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scalpels to cut vertically from the cartilage surface to the subchondral bone. After resection from the subchondral bone, the cartilage strips were weighed and frozen at −80 ◦ C or processed for chondrocyte isolation. Synovial fluid from OA patients with joint effusion was aseptically aspirated. Normal synovial fluid was obtained from organ and tissue donors who did not have detectable OA. The synovial fluid was centrifuged at 800 × g for 15 min, after which the supernatant was divided into 1 mL aliquots and frozen at −80 ◦ C. 2.2.1. Chondrocyte isolation and culture The cartilage tissue strips obtained as described above were digested with 2.5 mg/mL trypsin in DMEM at 37 ◦ C for 10 min. After removing the trypsin solution, the cartilage slices were incubated for 12–16 h with type IV clostridial collagenase (2 mg/mL) in Dulbecco’s modified Eagle’s medium (DMEM) with 5% FCS to release the cartilage cells. The chondrocytes were recovered and plated at high density (4 × 106 per 162-cm2 flask) in DMEM supplemented with 100 U/mL penicillin, 100 ␮g/mL streptomycin, 1% glutamine and 10% FCS. The cells were incubated at 37 ◦ C in a humidified gas mixture containing 5% CO2 balanced with air. Confluency was achieved at 2–3 weeks of primary culture. The cells were serumstarved in fresh DMEM with 0.5% FCS for 48 h prior to trypsinization. Cell viability was assessed by trypan blue dye exclusion. Finally, the chondrocytes (3–5 × 106 ) were recovered from the culture flasks by trypsinization, washed twice in a buffer containing 130 mM NaCl, 5 mM KCl, 2.5 mM Tris–HCl (pH 7.5) and 0.7 mM Na2 HPO4 , and transferred to microcentrifuge tubes for protein extraction. 2.2.2. Synoviocyte isolation and culture Synovial membranes were minced and exposed to enzymatic digestion with 1.25 mg/mL trypsin in PBS 1 h at 37 ◦ C with agitation. Dissociated cells were plated onto 162-cm2 flasks and cultured overnight in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 ␮g/mL streptomycin, 2 ␮M glutamine and 10% FCS at 37 ◦ C with 5% CO2 . Non-adherent cells were discarded and the adherent cells were cultured further in fresh medium until confluent, when the cells were trypsinized and subcultured. Synoviocytes from passages 4–8 were recovered from the culture

Fig. 1. Schematic workflow for the preparation of different protein samples obtained from human joints for two-dimensional electrophoresis (2-DE) analysis.

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flasks, washed twice, and transferred to microfuge tubes for protein extraction. 2.3. Cartilage glycosaminoglycan (GAG) precipitation We optimized our procedure for 2-DE of cartilage samples by precipitating their GAGs with cetylpyridinium chloride (CPC) with subsequent washing of the sample. Briefly, cartilage samples were pulverized at −80 ◦ C in a tissue grinder, and the resulting powder was transferred to a microfuge tube. The cartilage proteins were extracted using a mild extraction buffer composed of 500 mM NaCl, 50 mM HEPES, pH 7.2, and protease inhibitors. Extraction was carried out for 1 h at room temperature with agitation and occasional vortexing. The samples were cleared by centrifugation at 12,000 × g for 10 min; the supernatant was either stored at −80 ◦ C or subjected to proteoglycan (GAG) removal. The removal of GAGs was done using the method of Hermansson et al. [14], with some modifications. Briefly, the GAG content was determined using the dimethylmethylene blue assay [15], and GAGs were precipitated by incubation with 3 mg CPC per mg GAG. After precipitate removal, the proteins in the supernatant were precipitated overnight at 4 ◦ C with 1/10 vol. of 0.2% sodium deoxycholate (NaDOC) and 1/10 vol. of 100% trichloroacetic acid (TCA). The remaining CPC was removed from the precipitate by cationic exchange, washing the pellet with 1 mL 0.4 M sodium acetate in 90% ethanol for 30 min with gentle agitation. The pellets were washed with 1 mL 100% ethanol at −20 ◦ C to remove excess salts, and then with 100% acetone at −20 ◦ C prior to solubilization in ULB as described in the next section. 2.4. General procedures used for two-dimensional electrophoresis (2-DE) 2.4.1. Protein solubilization Tissue proteins or cell pellets were solubilized by vortexing followed by gentle agitation during a 1 h incubation in 200 ␮l of an isolectric focusing-compatible lysis buffer (ULB) containing 8.4 M urea, 2.4 M thiourea, 5% cholamidopropyl diethylamoniopropane sulfonate (CHAPS), 1% carrier ampholytes (IPG buffer pH 3–10 nonlinear (NL)), 0.4% Triton X-100 and 2 mM dithiothreitol (DTT). For protein quantification, 10 ␮l of the protein extract were diluted 10× with distilled deionized water and precipitated for at least 1 h using 0.02% sodium deoxycholate and 10% trichloroacetic acid. The precipitate was washed once with two volumes of ice-cold acetone, allowed to dry, and solubilized in alkaline sodium dodecyl sulphate (SDS) (5% SDS, 0.1 N NaOH). 5–10 ␮l of this sample were employed to quantify the proteins in each lysate using the bicinchoninic acid (BCA) technique (Pierce Chemical Company, Rockford, IL, USA). 2.4.2. First dimension: isoelectric focusing Solubilized protein (100 ␮g) was incubated for 1 h with gentle agitation in a rehydration buffer (8.4 M urea, 2 M thiourea, 2% CHAPS, 0.5% carrier ampholytes, 1.2% Destreak Reagent (GE healthcare) and 0.002% bromophenol blue). The proteins were then applied to 24 cm, pH 3–10 NL, IPG strips (GE Healthcare) using passive overnight rehydration. Focusing was performed at 20 ◦ C for a total of 64,000 Vhr (IPGphor, GE Healthcare). The strips were stored in tubes at −80 ◦ C. 2.4.3. Second dimension electrophoresis using SDS-PAGE Second dimension electrophoresis followed equilibration of the strips for 15 min in 6 M urea, 50 mM Tris–HCl (pH 8.8), 30% glycerol, 2% SDS and 1% DTT (reduction step), and 15 min in 6 M urea, 50 mM Tris–HCl (pH 8.8), 30% glycerol, 2% SDS, 0.002% bromophenol blue and 4% iodoacetamide (alkylation step). The strips were first washed with electrophoresis buffer (25 mM Tris–HCl,

pH 8.3, 192 mM glycine, 0.1% SDS) then attached to the surface of a 21 cm × 26 cm 10% polyacrylamide gel using 0.5% agarose. Electrophoresis was carried out according to the procedure of Laemmli [7], but using Tris–glycine electrophoresis buffer for the lower (anode) buffer and 2× Tris–glycine for the upper (cathode) buffer. Samples were run at 2.5 W/gel for the first 30 min and then at 17 W/gel until the bromophenol blue dye reached the bottom of the gel. 2.4.4. Gel staining Gels were stained with either Coomasie Blue or silver nitrate. Coomasie staining used 0.1% Brilliant Blue G-250 (Sigma–Aldrich) in 40% methanol with 10% acetic acid, followed by destaining in 40% methanol with 5% acetic acid. Silver nitrate staining was performed using standard protocols [8] with modifications. Briefly, gels were fixed overnight at 4 ◦ C in 40% ethanol with 10% acetic acid and washed three times for 10 min with water. Sensitization was carried out for 1 min in 0.02% sodium thiosulphate (Fluka, Buchs, Switzerland). After two consecutive 1-min water washes, the gels were impregnated with 0.2% silver nitrate (Fluka) in 0.075% formalin for 60–90 min. Excess silver nitrate was removed by a few seconds of water rinses, and stain development was carried out in a solution containing 3% potassium carbonate, 12.5 mg/l sodium thiosulphate and 0.025% formalin. Once the bands or spots on the gel reached the desired intensity, the reaction was stopped by transferring gels to 3% Trizma-base (Sigma–Aldrich) in 10% acetic acid. After 30 min in the stop solution, gels were scanned and dried or stored at 4 ◦ C in water. 2.4.5. Image acquisition Coomasie and silver-stained gels were digitized using a densitometer (ImageScanner, Amersham), and analyzed with PDQuest 7.3.0 computer software (Bio-Rad, Hercules, CA, USA). Utilizing PDQuest tools, protein spots were enumerated, quantified and characterized by their molecular mass and isoelectric point by bilinear interpolation between landmark features of each image previously calibrated with internal 2-DE standards (Bio-Rad). 2.5. Mass spectrometry identification of proteins 2.5.1. Protein in-gel digestion Selected spots of interest on the gel were excised and transferred to microcentrifuge tubes. Samples selected for analysis were reduced, alkylated and digested with trypsin according to Sechi and Chait [9]. Briefly, spots were washed twice with water, shrunk with 100% acetonitrile and dried in a Savant SpeedVac (Thermo Scientific, Waltham, MA, USA). The samples were then reduced with DTT and subsequently alkylated with iodoacetamide. Finally, samples were digested overnight at 37 ◦ C with 12.5 ng/␮l sequencing-grade trypsin (Roche Molecular Biochemicals, Indianapolis, IN, USA). 2.5.2. Mass spectrometry analysis After digestion, the supernatant was collected and 1 ␮l was spotted onto a MALDI target plate and allowed to air-dry at room temperature. Subsequently, 0.5 ␮l of a 3 mg/mL solution of ␣cyano-4-hydroxy-trans-cinnamic acid matrix (Sigma–Aldrich) in 0.1% TFA–50% acetonitrile (ACN) was applied to the dried peptide digest spots and again allowed to air-dry. The samples were analyzed using the MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA). MALDI-TOF spectra were acquired in reflector positive-ion mode at 1000 laser shots per spectrum. Mass spectra were internally calibrated using autoproteolytic trypsin fragments and externally calibrated using a standard peptide mixture (Sigma–Aldrich). TOF/TOF fragmentation spectra were acquired by selecting the 10

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most abundant ions of each MALDI-TOF peptide mass map, excluding trypsin autolytic peptides and other known background ions, at an average of 2000 laser shots per fragmentation spectrum. The parameters used to analyze the data were a signal-to-noise threshold of 20 and resolution higher than 10,000 with a mass accuracy of 20 ppm.

ized with Coomasie blue or mass spectrometry-compatible silver nitrate staining. Selected spots were then excised, destained, in-gel digested with trypsin and analyzed by MALDI-TOF/TOF mass spectrometry. The two-dimensional maps shown in the present work are representative of the results obtained in our laboratory. The proteomic patterns were found to be highly reproducible.

2.5.3. Database search The monoisotopic peptide mass fingerprinting data obtained from MS and the aminoacidic sequence tag obtained from each peptide fragmentation in MS/MS analyses were used to search for protein candidates using Mascot 1.9 (http://www.matrixscience.com). Tryptic autolytic fragments and other contaminations were removed from the dataset used for the database search. Search parameters for peptide mass fingerprints and tandem MS spectra were set using databases SWISS-PROT/TrEMBL (http://www.expasy.ch/sprot) and NCBI (http://www.ncbi.nlm.nih.gov). Fixed and variable modifications were considered (Cys as the S-carbamidomethyl derivate and Met as oxidized methionine, respectively) allowing one missed cleavage site, a precursor tolerance of 50 ppm and MS/MS fragments tolerance of 0.3 Da. The accepted isoelectric point range was from 3 to 10 and the accepted protein mass range was from 10 to 100 kDa. A positive identification was considered to be the achievement of at least five matching peptides and when at least 20% of the peptide coverage of the theoretical sequences matched, within a mass accuracy of 50 or 25 ppm with internal calibration. In all the positive identifications, the probability scores were greater than the score fixed as significant with a p value <0.05.

3.2. Pre-treatment and 2-DE of cartilage and synovial membrane samples

3. Results and discussion 3.1. Overview of sample preparation for proteomic analysis Fig. 1 shows the workflow we designed for the study of joint proteomes using 2-DE. The diverse protein samples were obtained following the procedures described in Section 2.2. Despite the differences between samples described below, the proteins were all extracted using a urea-based lysis buffer and most were cleaned with sodium deoxicholate-trichloroacetic acid precipitation. For the first dimension of the 2-DE analysis, protein extracts were separated with nonlinear immobilized pH gradient strips covering a wide pH range (3–10) to make possible the study of the complete proteome. In the second dimension, proteins were fractionated on large-format SDS-PAGE gels and visual-

3.2.1. Cartilage Articular cartilage is a tissue with a very low cell density and an extracellular matrix (ECM) composed mainly of water (approximately 75% of tissue weight) and an abundant network of collagen (predominantly type II), proteoglycans and other macromolecules. These features render cartilage proteomic analysis difficult because the relatively few structural proteins are in such abundance that other interesting components may remain masked and undetectable. A number of strategies for enrichment of the less abundant cartilage proteins and removal of contaminants have been developed. These include ultrafiltration [10,11], the use of differential protein solubilization procedures [12,13], and proteoglycan substraction by cetylpyridinium chloride precipitation [12,14] or CsCl gradient ultracentrifugation [13]. Moreover, proteoglycans are strongly anionic, which interferes with isoelectric focusing and makes their removal essential to obtain definitive 2DE gels. A selective loss or enrichment of some proteins may have occurred due to sample preparation in our studies; this should be considered when interpreting results. We optimized our procedure for 2-DE of cartilage samples by precipitating their GAGs with cetylpyridinium chloride (CPC) with subsequent washing of the sample (see Section 2.3 and Fig. 1). Fig. 2 shows 2-DE maps of human cartilage proteins obtained prior to (A) and after (B) GAG removal. The differences between the images illustrate the advantage of proteoglycan removal and the efficiency of this approach. The most representative spots from human cartilage maps were excised from the gels to identify the proteins shown in Fig. 3A and listed in Table 1. These included collagen and collagenassociated proteins, such as fibromodulin (FMOD) and cartilage oligomeric matrix protein (COMP). Interestingly, both FMOD and COMP have been proposed as markers for alterations in cartilage metabolism. Degraded fragments of FMOD have been found in RA and OA cartilage [16], increasing with age [17]. Elevated mRNA levels in human OA cartilage and increased total amounts of translated FMOD protein indicate that attempts to repair damaged cartilage

Fig. 2. Effect of glycosaminoglycan (GAG) removal on the proteome of cartilage. Two-dimensional electrophoresis (2-DE) gels of articular cartilage proteins before (A) and after (B) removal of GAGs by cetylpyridinium chloride (CPC) precipitation, showing the improvement in isoelectric focusing obtained after high anionic GAG depletion (IPG strip pH 3–10 NL, 10% T SDS-PAGE, silver staining).

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Fig. 3. Two-dimensional electrophoresis (2-DE) map of human articular cartilage (A) and synovial (B) proteins. Proteins are identified by the names used in the SWISS-PROT database (IPG strip pH 3–10 NL, 10% T SDS-PAGE, silver staining). Protein names are listed in Tables 1 and 2.

may be occurring [16,18]. COMP has received great interest in recent years as a biomarker for joint damage, including that resulting from RA [19], OA [20] and other skeletal diseases, as recently reviewed in Ref. [21]. 3.2.2. Synovial membrane The synovial membrane is located within the joint space and maintains normal joint function. Its anatomy can vary, but it often has two layers. The outer layer, or subintima, can be fibrous or fatty, whereas the inner layer, or intima, consists of a sheet of synoviocyte cells of two types, fibroblasts and macrophages. The fibroblast-like synoviocytes manufacture and secrete hyaluronan [22] and lubricin [23] into the synovial fluid, while the macrophages are responsible for the removal of undesirable substances from the synovial fluid. Following a procedure similar to that used for cartilage samples, synovial membranes from healthy joints were pulverized at −80 ◦ C and synovial proteins were extracted for 1 h using 1 mL of a non-ionic extraction buffer composed of 5 M urea, 1 M thiourea, 2% CHAPS and 2 mM DTT. The samples were then cleared by two successive centrifugations at 12,000 × g for 10 min; the supernatants

were placed overnight in NaDOC/TCA for protein precipitation, as previously described. Synovial samples are characterized by a high lipid content, requiring that the protein pellet be washed with 500 ␮l of 100% acetone at −20 ◦ C for 30 min to remove these lipids. The resulting pellet was then dried and solubilized in ULB as described in Section 2. Fig. 3B shows a representative 2-DE map of normal synovial proteins obtained by this technique. Table 2 lists some proteins indentified from this 2D map. In synovial membranes, we found an abundance of proteins related to fibrous structures, such as myosin and tropomyosin, in accordance with the described characteristics of the subintima. Fig. 3 shows the very different proteomic patterns obtained from cartilage and synovium, reflecting the diversity of the structures of the human joint. Proteomes of degenerative/inflamed synoviums from RA, OA and a chronic arthritic condition, spondyloarthropathy (SpA) were compared using 2-DE followed by tandem mass spectrometry [24]. A similar strategy of separating whole tissue proteins by 2-DE prior to western blotting with sera from RA patients was employed to detect novel citrullinated autoantigens of synovium in RA [25].

Table 1 Sixteen representative proteins from human normal articular joints identified by mass spectrometry of cartilage protein extracts separated by two-dimensional electrophoresis (2-DE). Spot namea

Description

Acces. No.a

Mr preb (kDa)

pI preb

No. of matched peptidesd

Seq. cov.e (%)

ACTB APOA1 COL6A1 COL6A2 COMP ENOA FMOD FMOD FRIL GDIR GSTP1 MIME PA2 PRDX2 SAMP VIME VIME

Actin beta chain Apolipoprotein A-I precursor (fragment) Collagen, type VI, alpha 1 precursor Alpha 2 type VI collagen, isoform 2C2 Cartilage oligomeric matrix protein Alpha enolase Fibromodulin precursor Fibromodulin Ferritin, light chain Rho gdp dissociation inhib. alpha, chain E Glutathione S-transferase P Mimecan (osteoglycin) Phospholipase A2 Peroxiredoxin 2 isoform b Serum amyloid P component, chain A Vimentin Vimentin

P60709 P02647 Q7Z645 Q6P0Q1 P49747 P06733 Q06828 Q8IV47 P02792 P52565 P09211 P20774 P04054 P32119 P02743 P08670 P08670

41.7 28.1 109.6 109.7 82.2 47.4 43.5 43.5 16.4 20.6 23.2 33.9 14.7 16 23.4 53.6 53.6

5.29 5.27 5.29 5.85 4.38 6.99 5.55 5.66 5.65 6.73 5.44 5.46 9.38 6.13 6.12 5.06 5.06

17 21 18 17 19 18 10 11 7 8 FQDGDLTLYQSNTILRf 8 7 8 10 29 15

58 64 21 18 34 45 20 28 32 35

Experimental Mr and pI calculated by analysis of the gel images with PDQuest 7.3.0 software. a Protein name and accession number according to SwissProt and TrEMBL databases. b Predicted Mr and pI according to protein sequence and Swiss-2DPAGE database. d Number of peptide masses matching the top hit from MS-Fit PMF. e Aminoacidic sequence coverage for the identified proteins. f Sequence tag identified by tandem mass spectrometry using MALDI-TOF/TOF MS.

30 52 39 38 59 37

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Table 2 Twenty-seven representative proteins from human normal articular joints identified by mass spectrometry of synovial membrane two-dimensional electrophoresis (2-DE). Spot namea

Description

Acces. No.a

Mr preb (kDa)

pI preb

No. of matched peptidesc

Seq. cov.d (%)

A1AT AACT ACTC ACTS APOA1 CAH1 CO3 CRYAB FETUA GPX3 HBB HBB HBD KCRM MLRV

Alpha-1-antitrypsin precursor Alpha-1-antichymotrypsin precursor Actin alpha cardiac Actin, alpha skeletal muscle Apolipoprotein A I precursor Carbonic anhydrase 1 Complement C3 precursor Alpha crystallin beta chain ␣-2-HS-glycoprotein precursor (Fetuin A) Glutathione peroxidase 3 precursor Hemoglobin beta chain Hemoglobin beta chain Hemoglobin delta chain Creatine kinase M-type Myosin regulatory light chain, ventricular/cardiac muscle isoform Myosin heavy chain, cardiac muscle ␣ Myosin heavy chain, cardiac muscle ␤ Myosin light polypeptide 3 Serum amyloid P-component precursor Tropomyosin 1 alpha chain Tropomyosin beta chain Tropomyosin alpha-3 chain Tropomyosin alpha-4 chain Serotransferrin precursor Zinc finger protein 622

P01009 P01011 P68032 P68133 P02647 P00915 P01024 P02511 P02765 P22352 P68871 P68871 P02042 P06732 P10916

46.9 47.8 42.3 42.4 30.8 28.8 187.2 20.1 40.1 25.8 16.0 16.0 16.0 43.3 18.6

5.37 5.33 5.23 5.23 5.56 6.63 6.02 6.76 5.43 8.20 6.81 6.81 7.97 6.77 4.92

22 12 10 13 26 14 21 15 9 11 11 14 6 15 10

55 38 36 42 67 67 18 69 29 44 79 84 39 42 62

P13533 P12883 P08590 P02743 P09493 P07951 P06753 P67936 P02787 Q969S3

223.7 223.1 22.0 25.5 32.7 32.9 32.8 28.5 79.3 54.8

5.60 5.63 5.03 6.10 4.69 4.66 4.68 4.67 6.81 5.80

31 38 15 9 9 14 12 10 36 13

17 21 74 30 29 38 30 32 47 27

MYH6 MYH7 MYL3 SAMP TPM1 TPM2 TPM3 TPM4 TRFE ZN622

Abbreviations as in Table 1.

3.3. Pre-treatment and 2-DE of chondrocyte and synoviocyte samples A straightforward approach to analyze the human joint is by proteomic analysis of joint cells. Joint cells are responsible for ECM synthesis and turnover; the global study of their proteins may provide valuable information about processes occurring in these tissues in joints. Working directly with cells for proteomic studies is in many ways more straightforward than using samples from other sources because most cellular proteins are easy to extract, their quantitative dynamic range is not extremely high, and they are easily freed from matrix components. Moreover, the culture of joint cells is a widely used method for increasing the quantity of proteins available for extraction. This is particularly useful for cells occurring in low numbers in joint tissue, such as chondrocytes, which represent less than 5% of cartilage weight. Cultures of articular chondrocytes isolated from various animal and human sources currently serve as useful models to study mechanisms controlling responses to growth factors and cytokines. Cultured chondrocytes also shed light on the mechanisms of pathogenesis and degenerative processes of some rheumatic diseases, such as OA. Using cells in primary culture, our group succeeded in partially delineating the proteome of human normal articular chondrocytes [26], generating a chondrocyte 2-DE reference map that at present encompasses almost 200 identified spots that correspond to more than 140 individual proteins. More recently, a similar strategy was used to develop a comparative proteomic analysis of OA chondrocytes employing fluorescent staining of the gels, bioinformatic analysis and identification of proteins varying in abundance [27]. Fibroblast-like synoviocytes, which are the characteristic cell type present in the synovial membrane, have proved to be useful models for the study of joint inflammatory disease, such as RA [28]. Synoviocytes obtained from RA patients have been analyzed by 2-DE, leading to the identification of 192 distinct proteins [29], many of which are known to be involved in cartilage physiology or pathophysiology. For proteomic analysis, joint cells must be first isolated from their tissues by enzymatic digestion of the ECM with trypsin and, in the case of cartilage, collagenase. Freshly isolated cells can be

used for protein extraction and analysis procedures with the caveat that some characteristics of the resulting proteomic profiles may be caused by cellular responses to the enzymatic treatment. Chondrocytes easily de-differentiate into fibroblasts and should be used during the first tissue culture passage while synovial cells are stable during further subculturing and may be trypsin-digested and collected after as many as 12–15 passages. The isolated cells are washed and their proteins extracted in a urea-based lysis buffer. Cellular protein extracts were resolved by 2-DE without need for further cleaning. Chondrocytes (Fig. 4A) and synoviocytes (Fig. 4B) show a marked similarity in their proteomes. The comparison of the synoviocyte map with reference maps allows identification of 82 different proteins involved in a broad range of biological functions. Some of the most abundant proteins appearing in the maps are vimentin, gelsolin or lamin A, filament proteins characteristic of adherent cells. These and 13 other protein forms common to both proteomic maps are depicted in Fig. 4 to illustrate their similarity. 3.4. Pre-treatment for 2-DE of synovial fluid samples Proteomics shows great promise for discovery of biomarkers to improve the diagnostic capability for a wide range of diseases. Biological markers are needed to better understand and characterize disease types, monitor disease status and progression, and response to current and new therapies. The search for candidates to serve as specific markers of joint damage for early diagnosis of disease is a major aim in rheumatology research. Blood plasma, serum, and other body fluids that come in contact with a variety of tissues are expected to be the best sources of protein biomarkers for proteomic analysis. These circulating fluids have recently been shown to be the most likely carriers of proteins secreted or shed by tissues [30]. The major advantage of serum is its ready availability, but proteins secreted or released from a specific tissue or cell type, i.e. those that hold the highest potential as biomarkers, are so diluted in blood that they are often undetectable by current methods. This has generated great interest in the analysis of “proximal” body fluids, which have been in contact with only one or a few tissues, so less dilution of tissue-derived proteins would be expected. Synovial fluid is one of these “proximal” fluids, being derived directly from the site of joint

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Fig. 4. Two-dimensional electrophoresis (2-DE) maps of human articular chondrocyte (A) and synoviocyte (B) proteins. Representative proteins are identified by the names used in the SWISS-PROT database (IPG strip pH 3–10 NL, 10% T SDS-PAGE, silver staining).

Fig. 5. Effect of albumin and immunoglobulin G (IgG) depletion on the synovial fluid proteome. (A) Whole human synovial fluid two-dimensional electrophoresis (2-DE) map. (B) Silver-stained SDS-PAGE gel showing the effect of depletion on the synovial fluid protein profile. Lane 1: non-depleted synovial fluid; lane 2: depleted synovial fluid; lane 3: protein fraction bound to the affinity resin. (C) 2-DE map of human synovial fluid proteins (IPG strip pH 3–10 NL, 10% T SDS-PAGE, silver staining). The 16 mapped proteins are identified by the names used in the SWISS-PROT database. Protein names are listed in Table 3.

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Table 3 Thirteen representative proteins from human osteoarthritic joints identified by mass spectrometry of two-dimensional electrophoresis (2-DE) gels of synovial fluids. Spot namea

Description

Acces. No.a

Mr preb (kDa)

pI preb

No. of matched peptidesc

Seq. cov.d (%)

A1AT A2MG AACT APOA1 CO3 FETUA HBB HBD HPT IGHG1 SAMP TRFE TTHY

Alpha-1-antitrypsin precursor Alpha-2-macroglobulin precursor Alpha-1-antichymotrypsin precursor Apolipoprotein A I precursor Complement C3 precursor ␣-2-HS-glycoprotein precursor (Fetuin A) Hemoglobin beta chain Hemoglobin delta chain Haptoglobin precursor Immunoglobulin gamma 1 chain C Serum amyloid P-component precursor Serotransferrin precursor Transthyretin precursor

P01009 P01023 P01011 P02647 P01024 P02765 P68871 P02042 P00738 P01857 P02743 P02787 P02766

46.9 163.3 47.8 30.8 18.9 40.1 16.0 16.0 45.9 37.0 25.5 79.3 16.0

5.37 6.00 5.33 5.56 6.02 5.43 6.81 7.97 6.13 8.46 6.10 6.81 5.52

24 22 12 26 20 9 14 6 14 7 9 36 7

56 21 38 67 15 29 84 39 32 33 30 47 69

Abbreviations as in Table 1.

disease. Proteomic studies in rheumatology have been designed to identify prospective biomarkers in SF, and attempt to then validate them in serum. SF is a hyaluronic acid-rich fluid secreted by the synovium. Under normal conditions it lubricates the joint and provides articular cartilage with the nutrients necessary for chondrocyte metabolism. It also serves as the intermediate carrier of proteins shed by articular cartilage and transferred to the systemic circulation. SF sampling is invasive, thus samples need to be centrifuged to remove contaminating cells, such as mononuclear cells before use for proteomics. It is also useful to digest samples with hyaluronidase to remove the HA, which may complicate proteomics analyses [31]. In both serum and SF, large quantitative variability among samples has been reported for a number of proteins when using 2-DE on samples from OA patients. One study found that 41 of 139 spots sampled showed differences of greater than threefold between any two samples, with nine having greater than 100-fold differences [32]. This study illustrates the need to analyze a large number of samples and a clearly defined reference map for normal SF proteins to differentiate between changes due to OA and normal variation between individuals. Variability among samples was also noted in a proteomic study of recurrent joint inflammation in juvenile idiopathic arthritis, where both plasma and synovial fluid were analyzed using 2-DE and cases with single-event knee joint inflammation were compared to a group with recurrent knee disease [33]. The large dynamic range of individual protein concentrations in plasma, serum, and synovial fluid is one of the most challenging issues in body fluid proteomics aimed at biomarker discovery. Disease biomarkers typically appear in low concentrations, making their detection difficult in the presence of proteins appearing in higher abundance. A practical and effective strategy solution for this problem is the removal of diagnostically uninformative high abundant proteins, thus enhancing the detection of low abundance proteins to penetrate deeper into the plasma proteome. Several systems to deplete plasma of the unwanted high concentration proteins have been developed and are commercially available. They use antibody-based resins with specific affinities for as many as 20 of the most abundant proteins in plasma. We employed resins with affinity for HSA, which represents about 65% of total plasma proteins, and IgG, which represents about 15% of total plasma proteins. Because SF and plasma have a similar composition of high abundance proteins this technique is also suitable for depleting SF of these proteins. Fig. 5A is a 2D map of synovial fluid showing the remarkably high concentrations of albumin and IgG. The affinity chromatography protocol to remove these two proteins is illustrated in Fig. 5B. A conventional SDS-PAGE gel was used to evaluate the protein profiles of SF before (lane 1) and after (lane 2) protein depletion, as well as to define the protein fraction bound to

Table 4 Principal protocol recommendations. Joint sample

Protocol modification before 2-DE

Cartilage

Glycosaminoglycan depletion by CPC precipitation. Lipid removal with acetone. Follow protocol for chondrocytes [26].

Synovium Cells (synoviocytes and chondrocytes) Synovial fluid

Depletion of albumin and IgG by affinity chromatography.

CPC: cetylpyridinium chloride.

the affinity resin (lane 3). Depletion of these two proteins enabled better visualization of other proteins (Fig. 5C), and improved identification of lower abundance proteins. Table 3 shows those less abundant proteins identified using this technique. 4. Conclusions The proteomic analysis of the articular joint tissues and cells should prove very helpful for increasing our insight into the endogenous control mechanisms of joint matrix turnover and unravelling the molecular and cellular mechanisms that participate in arthritic disease onset and progression. We have described a set of sample pre-treatment protocols that improve 2-DE proteomic analyses of cartilage, synovium, chondrocytes, synoviocytes, and synovial fluid. The main points of the optimized procedures are summarized in Table 4 and should prove useful to increase knowledge about joint pathogenesis. Hopefully, the proteomic analysis of synovial fluid and other body fluids will result in definition of biomarkers valuable for early diagnosis and monitoring of joint disease. Acknowledgements We are thankful to Mrs. Pilar Cal for her expert secretarial assistance. The authors express their appreciation to the Pathology Service and to Mrs. Lourdes Sanjurjo, and Mrs. Dolores Velo ˜ for providfrom the Orthopaedics Department of CHU A Coruna ing cartilage samples. This work was supported by grants from the Ministerio de Educación y Ciencia, Spain (SAF2005–06211), Fondo Investigación Sanitaria, Spain (CIBER-CB06/01/0040) and Secretaria I + D + i (PGIDIT06PXIB916358PR). C. Ruiz was supported by Programa Parga Pondal, Secretaria Xeral I + D + i, Xunta de Galicia. J. Mateos was supported by Fondo Investigación Sanitaria-Spain (CA07/00243). V. Carreira was supported by Xunta de Galicia (PGEIDIT06PXIC916175PN). Patricia Fernandez was supported by Fondo Investigación Sanitaria, Spain (CIBER-CB06/01/0040).

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