Abstract
Objective
Rheumatoid arthritis (RA) is an autoimmune disease that targets the synovium. The autoantigens involved in the autoantibody responses in RA are unknown. A targeted proteomics approach was used to identify proteins in RA synovial fluid (SF) that are recognized by autoantibodies in RA sera.
Methods
RA SF, depleted of abundant proteins, was fractionated by two-dimensional liquid chromatography (chromatofocusing followed by reverse phase HPLC). Protein arrays constructed from these fractions were probed with RA and normal control sera, and proteins within reactive fractions were identified by mass spectrometry. The reactivity of RA sera to an identified peptide was confirmed by ELISA.
Results
RA sera specifically reacted to a SF fraction containing fibrin. Mass spectrometry analyses established the presence of a citrullinated arginine at position 271 of the fibrin fragment present in RA SF. A synthetic peptide corresponding to fibrin residues 259–287, containing the citrulline substitution at Arg 271, was recognized by 10 of 12 RA sera, but by two of 18 normal control sera and one of 10 systemic lupus erythematosus sera.
Conclusion
Proteomics methodology can be used to directly characterize post-translational modifications in candidate autoantigens isolated from sites of disease activity. The finding that RA sera contain antibodies to the citrullinated fibrin 259–287 peptide may ultimately lead to improved diagnostic tests for RA and/or biomarkers for disease activity.
Similar content being viewed by others
Introduction
Rheumatoid arthritis (RA) is an autoimmune disease that targets the joints and affects 0.8% of the adults worldwide [1, 2]. Chronic joint inflammation leads to cartilage and bone destruction, resulting in loss of function. Many self-antigens have been implicated in the triggering and/or maintenance of autoreactive lymphocyte responses in RA [3–5]. Nevertheless, there remains an uncertainty as to how disease is caused and maintained [6]. Aberrant post-translational modifications of self-proteins may play a role in breaking T and B cell tolerance, leading to autoimmunity [7–9]. Of particular interest to the clinical management of RA are the anti-citrulline antibodies [10], which can predict both development [11, 12] and severity of disease [13, 14]. Citrullination is the post-translational modification (deimination) of arginine to citrulline catalyzed by protein arginyl deiminase (PAD) enzymes [15]. This conversion changes the charge of the site from a positive to a neutral and increases the mass of the amino acid by 1 Da. The difference in charge may cause protein unfolding [16], thereby exposing novel epitopes.
The current diagnostic test for anti-citrulline antibodies employs a cyclic citrullinated peptide (CCP), yet the citrulline residues on synovial joint proteins that are target(s) of anti-citrulline antibodies in vivo have not been precisely defined. Autoantigens, which exist in citrullinated forms include fibrinogen [17–19] (which was initially thought to be filaggrin [20, 21]), vimentin [22–24], collagen type I [25, 26], collagen type II [26–28], fibronectin [29], and alpha-enolase [30]. The presence of citrulline-modified fibrinogen alpha (FIBA) and beta chains in RA synovial tissue or fluid has been reported [17, 19].
The goal of this project was to develop a method to discover novel RA autoantigens using a targeted proteomic analysis. We and others have reasoned that autoantigens might be enriched in RA SF and specifically recognized by autoantibodies in RA sera. Previous autoantigen and biomarker discovery projects have employed one of several approaches to fractionate biological sample preparations, including two-dimensional PAGE, miniaturized chips with diverse surfaces to promote differential protein binding, and multidimensional LC–MS/MS [31]. Often these fractionation approaches were combined with immunoblotting with patient sera. Although they yielded some novel information, such methods were complicated by the wide dynamic range of protein concentrations in serum and SF, which obscures identification of potentially informative proteins in minor abundance.
To separate and probe SF proteins, we used a method that was previously used to characterize cancer antigens [32], which included two-dimensional liquid chromatography, protein arrays, and high-resolution mass spectrometry. Depletion of abundant serum proteins followed by protein fractionation via two-dimensional liquid chromatography increased the likelihood of identifying the lower abundance proteins. Protein arrays constructed from fractionated SF were probed with RA and control sera to identify biologically significant fractions, and further analyses were performed only on those targeted fractions. It is important to note that the arrays were constructed from clinical samples that contain proteins with their post-translational modifications acquired in vivo. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was then used to identify immunogenic protein fragments and their post-translational modifications. Through this targeted proteomics approach, we have identified a citrulline-modified Arg 271 residue, within a fibrin alphaC domain fragment stably present in RA SF, as a target of autoantibodies in RA sera.
Materials and Methods
Patient Samples
All samples were obtained using IRB approved protocols and all patients consented to be part of the study. SF and sera were obtained from patients receiving care in the outpatient Rheumatology clinics at the Los Angeles County and University of Southern California Medical Center. RA and systemic lupus erythematosus (SLE) patients were diagnosed according to established clinical criteria [33, 34]. All patients had to have a clinical diagnosis of RA as defined by rheumatologists at academic center. The sampling was on consecutive RA patients from LA County hospital who had a joint effusion that was aspirated. Due to use of biologic agents, much less effusions are seen in the clinic. Patient population was mostly Hispanic, approximately 80%, with active RA stage 2 to 4 with most probably falling into 2 or 3 but data not collected so this is speculative. Comorbidities were not examined. All samples processed within 4 h. Control sera were obtained from healthy volunteers at the City of Hope General Clinical Research Center. SF samples were diluted 1:5 in PBS, centrifuged to remove cellular debris, and stored at −80°C. There are no viscosity issues when the SF is diluted 1:5 in PBS. Blood samples were allowed to clot overnight at 4°C. The next day, the blood was centrifuged and the top layer of serum was transferred into new tubes. Serum samples were stored at −80°C until use.
Protein Fractionation
A multiple affinity removal column (Agilent Technologies, Wilmington, DE, USA) was used to remove six abundant proteins (albumin, IgG, antitrypsin, IgA, transferrin, and haptoglobin) from SF. The procedure removed 85–90% of the total protein mass, which increased the probability of detecting the lower abundance proteins. Depletion was performed according to manufacturer’s protocol. After the depletion, samples were desalted by use of a 5-kDa MWCO spin filter (Amicon Ultra-15, Millipore Corp., Bedford, MA, USA). Reactivity of RA sera to proteins in the 5-kDa filtrate was not detected. Protein concentration of the desalted SF was determined by RCDC protein assay (BioRad Laboratories, Hercules, CA, USA).
SF proteins were separated by 2D-HPLC, chromatofocusing, and reverse phase (RP) HPLC. A Beckman PF2D System (Beckman Coulter, Inc., Fullerton, CA, USA) with a PF2D kit (column and buffers) was used for the first-dimension separation. Approximately 5 mg of proteins was separated in the pH range 8.5 to 4.0. After loading, the sample was washed in start buffer for 20 min, eluant buffer for 75 min, and then 1 M sodium chloride buffer for 45 min. The column was then washed overnight in water. Fractions were collected in increments of 5 min or 0.2 pH units, whichever came first, into a cooled deep well 96-well plate. This separation was reproducible as performed according to manufacturers’ supplied protocol. The second-dimension separation was performed on a Vydac C4 column (5 um, 300 A, 2.1 × 250 mm) using the following program: hold for 12 min at 5% buffer B; 5% to 95% buffer B in 25 min; hold at 95% buffer B for 8 min. The flow rate was 0.25 ml/min. Fractions were collected between 14 and 45 min at every 2 min into standard 96-well plates using a fraction collector.
The pooled SF second-dimension separations were performed on a Vydac C4 column (10 um, 300 A, 4.6 × 250 mm) using a gradient of 2% to 98% buffer B in 60 min and a flow rate of 1 ml/min. Corresponding fractions that eluted in the same pH range from four first-dimension runs were combined to ensure enough material was obtained for analysis by mass spectrometry. Buffer A consisted of 0.1% TFA. Buffer B consisted of 0.1% TFA in 90% acetonitrile. A Beckman System Gold 126 equipped with a model 168 diode array detector (Beckman Coulter, Inc.) was used to perform the separations. Fractions were collected every minute. Samples were frozen and lyophilized to dryness.
Protein Arrays
Custom arrays were hand made using a VP 409 replicator with 96 pins each holding 100 nl of fluid (V&P Scientific, Inc., San Diego, CA, USA). The selected HPLC fractions were resuspended in ∼200 μl of 6 M urea/sodium bicarbonate pH 8.0. The microtiter plates were slowly rocked for about 30 min to facilitate protein solubility. The replicator was dipped into a 96-well plate, and the fluid (∼100 nl) retained on the tips of the pins was transferred to a nitrocellulose membrane. Each fraction was arrayed in triplicate. To serve as a positive control for serum antibody reactivity, an influenza vaccine preparation also was arrayed. After drying overnight in a laminar flow hood, the arrayed membranes were blocked overnight in a non-fat dried milk solution and subsequently were rinsed twice in TBS (20 mM Tris–HCl, 500 mM NaCl, pH 7.5). The arrays were incubated with a 1:200 dilution of RA or control sera for 1 h at room temperature. After rinsing twice in TBS, the arrays were incubated with a 1:100,000 dilution of HRP-conjugated F(ab′)2 goat anti-human IgG + IgM + IgA secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The arrays were washed twice with 3% newborn calf serum/0.05% Tween/TBS, twice with a 0.05% Tween/TBS, twice with TBS, and twice with water. ECL Plus Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA) were used as the detection agent. The arrays were imaged on a Typhoon 9410 (GE Healthcare) using the following conditions: Laser (457 nm), Emission filter (520BP40), Focal Plane (Platen), Pixel Size (50 μm), Sensitivity (Normal). Different voltages were used to scan the images but typically 500v was used for most of the images. To distinguish background noise from foreground signal by a statistical method that complemented the visual inspection of the protein arrays, the median filter smoothing technique was applied to the imaged signals as described [35].
Mass Spectrometry
Approximately 80% of the solubilized RP-HPLC fraction was digested with trypsin (Promega, Madison, WI, USA). Approximately 5% of the digested material was analyzed by LC/MS/MS. Analyses were performed on a Thermo Finnigan LTQ-FT linear ion trap-Fourier transform mass spectrometer (Thermo Electron Corporation, San Jose, CA, USA) coupled to an Eksigent nanoLC-2D capillary HPLC system (Eksigent Technologies, LLC, Dublin, CA, USA). Samples were loaded onto a 300 μm × 5 mm C18 trapping column (Dionex Corporation, Sunnyvale, CA, USA) and then eluted through a lab-built 75 μm × 10 cm analytical column packed with 3 μm C18 Pursuit resin (Varian, Inc., Palo Alto, CA, USA). The gradient for the trapping column was 100% A for 5 min using a flow rate of 10 μl/min. The gradient for the analytical column was 2% to 35% B in 45 min, 35% to 50% B in 4 min, and 50% to 95% B in 2 min using a flow rate of 0.2 μl/min. High-resolution full-scale mass spectra were acquired in the Fourier transform-ion cyclotron resonance (FT-ICR) section of the mass spectrometer while fragment ion (MS/MS) spectra were obtained from the linear ion trap section. Fragmentation was performed using a collision energy setting of 35. Dynamic exclusion was set at 15 s.
Monoisotopic peaks and peptide charge states were determined during acquisition by the Xcalibur acquisition software using the high-resolution Fourier transform mass spectrometry (FTMS) spectra. Protein identifications were made by SEQUEST [36]. SEQUEST searches were performed with the following parameters: use of the 10/17/08 release of the SwissProt database (downloaded from ftp://ftp.ncbi.nlm.nih.gov/blast/db), monoisotopic masses, partial trypsin cleavage, 2 amu peptide and fragment tolerance, and automatic charge state determination. The SwissProt database was filtered to include only entries containing _HUMAN as a parameter. Peptide hits were filtered using the following criteria: DeltaCn greater than or equal to 0.08, XC greater than or equal to 1.8 for peptides having +1 charge, 2.5 for peptides with a +2 charge, and 3.5 for peptides having a +3 charge and the peptide must be to a protein with probability score less than 0.0001 used [37]. Peptides meeting these criteria were further analyzed. Although the LTQ-FT has routine mass accuracy of 2 ppm, it is set to perform MS/MS on the most abundant peak in an isotope envelope, rather than the monoisotopic mass. This frequency results in errors of 1 or 2 mass units for large peptides. Because SEQUEST and extract_msn do not correct for this error, it was necessary to set peptide mass tolerance to 2.5 to account for errors in precursor assignment. Fragment ion tolerance was set at 0.0. The searches were performed assuming both trypsin and no enzyme specificity. Spectra were hand sorted to identify and verify post-translational modifications (PTM). All charge states and mass were manually verified using the high-resolution FTMS data. All spectra corresponding to candidate autoantigens were manually verified.
Synthetic Peptides
The FIBA 259–287 peptides with and without the citrulline substitution for arginine 271 (MELERPGGNEITR*GGSTSYGTGSETESPR) were synthesized in the City of Hope Peptide Synthesis Core Facility. The synthesized profilaggrin 619–631 peptide (SSFSQDR*DSQAQS) contained a citrulline for arginine substitution at position 625. Peptides were dissolved in 0.1% TFA and purified two to three times on a C18 reverse phase column. Concentration was determined by amino acid analysis.
ELISA
Maleic anhydride coated ELISA plates (Pierce Biotechnology, Inc., Rockford, IL, USA) were incubated overnight with 100 μl of 12.5 μg/ml peptide in PBS. The plates were blocked overnight in a dried milk solution, washed twice in TBS, and incubated overnight in a 1:20 dilution of patient sera. After rinsing two times in TBS, the plates were incubated in a 1:10,000 dilution of HRP-conjugated F(ab′)2 goat anti-human IgG + IgM + IgA secondary antibodies for 1 h. The plates were then rinsed 6 times in TBS. For color development 150 μl ABTS (Pierce Biotechnology) was added to the wells and incubated for 30 min. To stop the reaction, 100 μl of 1% SDS was added. The absorbance at 405 nm was read on a plate reader.
Results
A targeted proteomic approach was performed to identify autoantigens in RA SF. The experimental design is diagrammed in Fig. 1. An immunodepletion column was first used to remove six abundant serum proteins (albumin, antitrypsin, haptoglobin, IgA, IgG, and transferrin). The depleted SF was then fractionated by chromatofocusing HPLC (provided as Supplement Fig. 1) and reverse phase HPLC (provided as Supplement Fig. 2). This protein fractionation strategy increased the chance of identifying the lower abundance proteins by separating them from the higher abundance proteins. The second-dimension fractions were used to construct protein arrays on nitrocellulose membranes, which were used to test for the presence of autoantigens by analyzing differential reactivity of RA and control sera. We focused on one second-dimension fraction resulting from the separation of the first-dimension fraction eluting at pH 5.63–5.45 (Fig. 2) that tested positive when probed with RA serum but negative when probed with normal control serum. This was the only fraction that tested positive in which a peak was detected on the HPLC chromatographs. Although there was sufficient material for the protein array analysis, the mass spectrometry analysis did not yield a protein identification. Both the protein arrays and HPLC chromatographs appear to be more sensitive at detecting proteins than the mass spectrometry.
In order to obtain enough material for mass spectrometry analysis, SF from nine different patients was pooled (total volume of 50 ml) and used for a large-scale procedure. After protein depletion, approximately 20 mg of depleted SF material was used to perform four first-dimension chromatofocusing runs (Fig. 3a). The fractions that eluted at pH 5.63–5.45 were combined to perform one second-dimension RP-HPLC run (Fig. 3b). Fractions corresponding to the region that tested positive for RA serum binding in the protein array were digested with trypsin and analyzed by LC/MS/MS. SEQUEST searches were performed using the SwissProt database limiting the search to tryptic peptides. Table 1 lists the proteins found in these fractions and the corresponding peptides are included as supplementary material (Supplement Table 1).
a Representative HPLC chromatogram for the first-dimension chromatofocusing separation of the abundant protein depleted SF sample. The diagonal line indicates the observed pH gradient. The fractions corresponding to the elution range of pH 5.63–5.45 from four runs were combined for the second-dimension separation. b HPLC chromatogram for the second-dimension reverse phase separation. This sample was separated on a Beckman System Gold HPLC coupled to a diode array detector. The diagonal line indicates the solvent gradient (%B). The fraction (between 20 and 25 min) corresponding to the region that tested positive in the protein array assay was selected for further characterization by mass spectrometry
The candidate autoantigen specifically detected by the RA serum was estimated to be in fractions 20–22. Amongst other proteins, these fractions contained fibrinogen, a known autoantigen that can be citrullinated in vivo. Fibrinogen alpha (FIBA_HUMAN, SwissProt Accession # P02671) was identified in fraction 20 with nine unique peptide hits (15% sequence coverage) and in fraction 22 with 18 unique peptides (24% sequence coverage). Altogether, peptides were only found originating from the center of the FIBA protein (amino acids 250–599) corresponding to the alphaC domain of fibrin (amino acids 239–629). Figure 4 shows the sequence of the fibrin alphaC domain with the amino acids identified by mass spectrometry in bold and arrows pointing to any modifications. Peptides from the center region were not identified due a lysine-/arginine-rich area followed by two cross-linked amino acids (Ser461–Ser491).
Careful examination of the mass spectra assigned to arginine-containing FIBA peptides led to the assignment of a citrullinated peptide corresponding to FIBA 259–287. The calculated mass obtained from the mass spectrum (Fig. 5) was 1 Da higher than the calculated mass for the unmodified peptide. The lower resolution MS/MS spectrum obtained with the ion trap part of the LTQ-FT did not allow determination of the exact location of the modification. However, a number of other fragments corresponding to parts of the peptide (Table 2) were observed in the spectra obtained with the ion cyclotron resonance (ICR) analyzer of the LTQ-FT, which provided precise mass measurements. There are four sites on the FIBA 259–287 peptide that could possibly be modified resulting in a mass shift of +1 Da. The peptide contains three arginines that can be citrullinated and an asparagine that can be deaminated to form aspartic acid (Fig. 5). All fragments that did not show the expected tryptic cleavage at Arg 271 showed observed masses that were 1 Da higher than the expected mass for the unmodified form. This is consistent with the known failure of trypsin to cleave citrullinated Arg residues. Thus, only conversion of Arg 271 to citrulline is consistent with observed mass values of this FIBA fragment in RA SF and the failure of trypsin to cleave at that site.
As a final proof that the citrullination site was correctly assigned, the peptide corresponding to residues 259–287 was synthesized with and without the citrulline in position 271 (designated 271X and 271R, respectively). Both the charge state distribution in the electrospray spectrum and the fragment masses in the MS/MS spectrum of the 3+ charge state of 271X matched spectral data obtained with the sample isolated from RA SF.
To establish that the citrullinated FIBA 259–287 peptide was recognized specifically by RA patient sera, the two FIBA 259–287 synthetic peptides were tested in an ELISA. An additional citrullinated synthetic peptide, corresponding to profilaggrin 619–631 (FIL) with a citrulline substitution at Arg 625, was included as a control. The immobilized peptides were incubated with sera from RA, SLE, or healthy controls, followed by detection of bound antibodies by HRP-conjugated anti-human IgG, IgA, and IgM antibodies and a colorimetric assay. Graphs depicting the ELISA optical density readings are shown in Fig. 6. Of 18 healthy control sera tested, two reacted to the 271R peptide, two reacted to the 271X peptide, and one reacted to the FIL peptide. Of the 12 RA sera tested, four reacted to the 271R peptide, 10 reacted to the 271X peptide, and three reacted to the FIL peptide. Of the 10 SLE sera tested, one patient reacted to all three peptides. The number of sera that reacted exclusively to the 271X peptide, and not with the 271R nor FIL peptides, was 5/12 RA sera, 0/18 healthy sera, and 0/10 SLE sera. These results provide evidence that antibodies in a subset of RA sera bind specifically to the citrulline residue at position 271 of the FIBA alphaC domain found in RA SF.
RA sera contain antibodies that specifically bind FIBA peptides bearing a citrulline substitution at Arg 271. In an ELISA, RA, SLE or control (C) sera were incubated with plate-immobilized peptides corresponding to FIBA 259–287 with (271X) or without (271R) a citrulline at position 271, or to filaggrin 619–631 (fil) with a citrulline at position 625. Sera that reacted with 271R include: two out of 18 normals, four out of 12 RA, one out of 10 SLE. Sera that reacted with 271X include: two out of 18 normals, 10 out of 12 RA, one out of 10 SLE. Sera that reacted with fil include: one out of 18 normals, three out of 12 RA, one out of 10 SLE
Discussion
Biomarker analysis consists of three phases: discovery, verification, and validation [38, 39]. Discovery is performed by a thorough analysis of a few samples with hopes of identifying many candidate biomarkers to be used for further study. Verification is performed by determining the presence of select candidate biomarker in a large number of clinical samples to estimate sensitivity and specificity. Sensitivity is defined as the percentage of patients with the specified disease that test positive for the biomarker. Specificity is defined as the percentage of people that do not have the specified disease that test negative for the biomarker. Candidate biomarkers that have a high sensitivity and high specificity are further analyzed in the validation phase. Validation is performed by analyzing the presence of the candidates in a patient pool that is expected to be present in a clinical setting where patients with the specific disease will be diagnosed. The purpose of validation is to test whether the candidate can be used for diagnosis. After validation, the biomarkers may be used to develop a diagnostic test. This study represents the discovery phase of biomarker analysis.
We have used a proteomic method to fractionate RA SF proteins, determine their reactivity to autoantibodies in RA sera, and identify immunogenic antibody epitopes. This method involves depletion of abundant serum proteins, two-dimensional liquid chromatography, protein macroarrays probed with RA and control sera to identify fractions containing potential autoantigens, and mass spectrometric analyses of those fractions via high-resolution LC–MS/MS. We identified a portion of a SF protein, the fibrin alphaC domain fragment, whose immunogenicity depended upon an Arg to citrulline post-translational modification that had occurred in vivo. We have shown that RA autoantibodies specifically target an epitope containing citrulline at position 271 of FIBA and that this post-translational modification of Arg 271 is present in RA SF. This approach is a feasible strategy that can be used to identify or confirm other autoantigens in RA SF as well as self-proteins that are targets of autoreactive B cell responses in other autoimmune diseases.
Citrullinated fibrinogen is a known autoantigen in RA [17, 40]. Experiments using purified fibrinogen and PAD enzymes in vitro identified 22 possible citrullination sites in FIBA [41, 42]; the Arg 271 identified in our study was citrullinated by both PADI4 and PADI2 enzymes in vitro. Several groups have reported reactivity of RA sera to synthetic FIBA peptides [19, 43, 44] and fibrinogen present in synovial exosomes [18]. Another report showed that mAbs specific for the same citrullinated FIBA peptide identified in our study detected the peptide epitope’s presence in RA SF but not RA plasma [19]. The results from our study confirm the presence and reactivity of citrullinated fibrin/fibrinogen in SF. If our study had been performed using RA plasma as the source of autoantigens, we would not have been successful at identifying autoantigens. Identifying the best biomarker source is crucial in biomarker discovery.
It is possible that our study failed to detect additional immunogenic citrulline sites on FIBA as well as other molecules. The failure of trypsin to cleave at citrulline residues often results in large peptides that are difficult to characterize by mass spectrometry. Other autoantigens could be present at very low concentrations, lost due to non-specific binding during chromatography, did not bind to the nitrocellulose, did not bind in the right conformation to the nitrocellulose, are heavily modified or glycosylated, were undetected by the antibodies in the macroarrays, and/or were not completely digested by trypsin. For these and many more reasons, the proteins could have escaped detection by the macroarray or mass spectrometry experiments. This study was designed to identify proteins so if the autoantigens are carbohydrate, lipid, or another type of molecule then they would have also been undetected. The chromatographs are more sensitive at detecting proteins than the mass spectrometry. However, since small chromatography peaks yielded no protein identifications, the nature of the molecule that produced the peak is unknown.
The role for the fibrin alphaC domain fragment in RA pathogenesis may be complex. Soluble citrullinated fibrinogen and fibrin degradation products have been found in RA SF but not RA plasma, suggesting that there are high levels of active PAD enzymes in RA SF [19, 45]. Fibrin deposits in the joints of RA patients are widely observed and have been hypothesized to be the cause of pannus formation [46]. The fibrin deposits in the joints allow the fibrin molecules to remain in an inflammatory environment for a prolonged period of time, which could facilitate post-translational citrulline modification [5]. The alphaC domain fragments are the first to be released during fibrinolysis so they are constantly being generated [47]. If stably present, such degraded alphaC fragments could readily be taken up by antigen presenting cells and chronically displayed to the immune system. Hence, a degradation product of an aberrantly modified self-protein may be an RA autoantigen.
This discovery of post-translationally modified immunogenic epitopes present on self-proteins in vivo may contribute to improved diagnostic tests for RA. With the data obtained from a limited number of patients, the citrullinated 271X peptide seems to have similar sensitivity (83.3%) as the commercially available CCP2 test. Since antibodies to CCP have been shown to be present prior to disease-onset development [11, 12], there is a possibility that the citrullinated 271X peptide can also predict disease. Studies determining when autoantibodies to the citrullinated 271X are produced need to be conducted. Whether a test using the native peptide offers any improvement over the commercially available tests remains to be shown. Irreversible joint damage can occur early in the disease process [48], so early diagnosis and aggressive treatment is vital to the preservation of joint function. Autoantibodies specific for citrullinated epitopes are predominant in early RA patients with high-grade joint inflammation and clinical manifestations predicting development of severe erosive disease [43, 49]. One promising diagnostic tool to define clinically distinct subsets of RA patients is antigen microarray profiling of autoantibodies, an assay in which known autoantigens are arrayed on slides, which are probed with patient sera [43]. The method described here, which can be used to identify epitopes on proteins that are modified in inflamed synovial tissue in situ, will lead to additional information about autoantigens that will help to increase the power of such diagnostic autoantigen arrays.
In summary, proteomic analysis defined an immunogenic citrulline-containing epitope, within the fibrin alphaC domain fragment, as an autoantigen present in RA SF. This study provides further validation that citrullinated fibrinogen is an autoantigen in RA. The strategy used in this project should be useful for identification of novel autoantigens in RA and other autoimmune diseases.
References
Feldmann M, Brennan FM, Maini RN. Rheumatoid arthritis. Cell. 1996;85:307–10.
Firestein GS. Evolving concepts of rheumatoid arthritis. Nature. 2003;423(6937):356–61.
Corrigall VM, Panayi GS. Autoantigens and immune pathways in rheumatoid arthritis. Crit Rev Immunol. 2002;22(4):281–93.
Boekel MAv, et al. Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Rheum. 2002;4:87–93.
Gaalen Fv, et al. The devil in the details: the emerging role of anticitrulline autoimmunity in rheumatoid arthritis. J Immunol. 2005;175:5575–80.
Firestein G. Immunologic mechanisms in the pathogenesis of rheumatoid arthritis. J Clin Rheumatol. 2005;11:S39–44.
Utz PJ, Gensler TJ, Anderson P. Death, autoantigen modifications, and tolerance. Arthritis Res Ther. 2000;2:101–14.
Vossenaar ER, Venrooij WJv. Citrullinated proteins: sparks that may ignite the fire in rheumatoid arthritis. Arthritis Res Ther. 2004;6:107–11.
Plotz P. The autoantibody repertoire: searching for order. Nat Rev Immunol. 2003;3:73–8.
Schellekens GA, et al. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J Clin Invest. 1998;101:273–81.
Rantapää-Dahlqvist S, et al. Antibodies against cyclic citrullinated peptide and iga rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum. 2003;48(10):2741–9.
Nielen MMJ, et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis. Arthritis Rheum. 2004;50(2):380–6.
Lundberg K, et al. Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity. Arthritis Res Ther. 2005;7:R458–67.
Mil AHvdH-v, et al. Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis. Arthritis Res Ther. 2005;7:R949–58.
Suzuki A, Yamada R, Chang X, Tokuhiro S, et al. Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet. 2003;34:395–402.
Tarcsa E, et al. Protein unfolding by peptidylarginine deiminase. J Biol Chem. 1996;271(48):30709–16.
Masson-Bessière C, et al. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the a- and b-chains of fibrin. J Immunol. 2001;166:4177–84.
Skriner K, et al. Association of citrullinated proteins with synovial exosomes. Arthritis Rheum. 2006;54(12):3809–14.
Takizawa Y, et al. Citrullinated fibrinogen detected as a soluble citrullinated autoantigen in rheumatoid arthritis synovial fluids. Ann Rheum Dis. 2006;65:1013–20.
Girbal-Neuhauser E, et al. The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (Pro) filaggrin by deimination of arginine residues. J Immunol. 1999;162:585–94.
Sebbag M, et al. The antiperinuclear factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific autoantobodies. J Clin Invest. 1995;95:2672–9.
Vossenaar ER, et al. Rheumatoid arthritis specific anti-Sa antibodies target citrullinated vimentin. Arthritis Res Ther. 2004;6:R142–50.
Bang H, et al. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis Rheum. 2007;56(8):2503–11.
Tilleman K, et al. Synovial detection and autoantibody reactivity of processed citrullinated isoforms of vimentin in inflammatory arthritides. Rheumatology (Oxford). 2008;47(5):597–604.
Suzuki A, et al. Anti-citrullinated collagen type I antibody is a target of autoimmunity in rheumatoid arthritis. Biochem Biophys Res Commun. 2005;333:418–26.
Koivula MK, et al. Sensitivity and specificity of autoantibodies binding to citrullinated carboxyterminal telopeptides of types I and II collagens in an early arthritis series. Rheumatology (Oxford). 2008;47(5):656–9.
Burkhardt H, et al. Humoral immune response to citrullinated collagen type II determinants in early rheumatoid arthritis. Eur J Immunol. 2005;35:1643–52.
Yoshida M, et al. Autoimmunity to citrullinated type II collagen in rheumatoid arthritis. Mod Rheumatol. 2006;16(5):276–81.
Chang X, et al. Citrullination of fibronectin in rheumatoid arthritis synovial tissue. Rheumatology. 2005;44(11):1374–1382.
Kinloch A, et al. Identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis. Arthritis Res Ther. 2005;7:R1421–9.
Hueber W, Robinson WH. Proteomic biomarkers for autoimmune disease. Proteomics. 2006;6:4100–4115.
Yan F, et al. Protein microarrays using liquid phase fractionation of cell lysates. Proteomics. 2003;3:1228–35.
Arnett FC, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31(3):315–24.
Hochberg MC, et al. The American College of Rheumatology 1991 revised criteria for the classification of global functional status in rheumatoid arthritis. Arthritis Rheum. 1992;35(5):498–502.
Smith DD, et al. Median filter algorithm for estimating the threshold of detection on custom protein arrays. Biotechniques. 2006;41:74–8.
Yates JRd, Eat J, McCormack AL, Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem. 1995;67:1426–36.
Chen EI, et al. Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT). Mol Cell Proteomics. 2006;5:53–6.
Rifai N, Gillette MA, Carr SA. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol. 2006;24(8):971–83.
Zolg W. The proteomic search for diagnostic biomarkers: lost in translation? Mol Cell Proteomics. 2006;5(10):1720–6.
Chang X, Yamada R, Suzuki A, Sawada T, Yoshino S, Tokuhiro S, et al. Localization of peptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue of rheumatoid arthritis. Rheumatology (Oxford). 2005;44(1):40–50.
Nakayama-Hamada M, et al. Comparison of enzymatic properties between hPADI2 and hPADI4. Biochem Biophys Res Commun. 2005;327:192–200.
Kubota K, Yoneyama-Takazawa T, Ichikawa K. Determination of sites citrullinated by peptidylarginine deiminase using 18O stable isotope labeling and mass spectrometry. Rapid Commun Mass Spectrom. 2005;19:683–8.
Hueber W, et al. Antigen microarray profiling of autoantibodies in rheumatoid arthritis. Arthritis Rheum. 2005;52(9):2645–55.
Sebbag M, et al. Epitopes of human fibrin recognized by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins. Eur J Immunol. 2006;36:2250–63.
Sinz A, et al. Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. Electrophoresis. 2002;23:3445–56.
Sánchez-Pernaute O, et al. A fibrin based model for rheumatoid synovitis. Ann Rheum Dis. 2003;62:1135–8.
Henschen-Edman AH. Fibrinogen non-inherited heterogeneity and its relationship to function in health and disease. Annals NY Acad Sci. 2001;936:580–93.
Scott D. Radiological progression in established rheumatoid arthritis. J Rheumatol Suppl. 2004;69:55–65.
Gaalen FAv, et al. Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis. Arthritis Rheum. 2004;50(3):709–15.
Acknowledgements
The authors would like to thank Drs. Andrew J. Alpert, Alex Kurosky, Paul Plotz, and Henry M. Fales for reviewing the manuscript, Dr. Robert Nathan for discussions on array analysis, Dr. Leonid Medved for providing the structure of the alphaC domain, and the patients and volunteers who donated the serum and SF samples.
Author information
Authors and Affiliations
Corresponding author
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Supplement Table 1
Proteins/peptides identified in selected B2 second-dimension RP-HPLC fractions (PDF 1.25 mb)
Supplement Fig. 1
First-dimension chromatofocusing runs. Chromatographs depict separations of early RA SF (A), late RA SF (B), and serum from healthy control (C) (PDF 203 kb)
Supplement Fig. 2
Overlay of second-dimension RP-HPLC chromatographs of late RA SF (purple), early RA SF (blue), and healthy control serum (red) (PDF 1.5 mb)
Rights and permissions
Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
About this article
Cite this article
Cano, L., Arkfeld, D.G. Targeted Synovial Fluid Proteomics for Biomarker Discovery in Rheumatoid Arthritis. Clin Proteom 5, 75–102 (2009). https://doi.org/10.1007/s12014-009-9028-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12014-009-9028-1