Online data supplement (1486 words)

Serum samples

Clinical, biological and immunological parameters were recorded by use of a standardized form as detailed. Clinical parameters included skin involvement, vascular manifestations (pitting scars, digital ulcers, digital infarcts, scleroderma renal crisis, PAH), and pulmonary involvement (ILD, results of pulmonary function tests for forced vital capacity, total lung capacity and diffusion capacity for carbon monoxide [DLCO]). PAH was detected by echocardiography, with a tricuspid gradient of 30 mmHg (i.e., an estimated systolic pulmonary arterial pressure  30+10=40 mmHg assuming a right atrial pressure of 10 mmHg in all patients) [1], and in 14 out of the 32 patients with SSc-PAH, PAH was confirmed by RHC.Scleroderma renal crisis was defined as rapidly progressive oliguric renal insufficiency with no other explanation and/or rapidly progressive hypertension during the course of SSc.

Biological and immunological data were collected on anti-nuclear Abs, anti-centromere Abs, and anti-extractable nuclear antigens Abs.

Detection of antibody reactivity with cell antigens using 2-DE and immunoblotting

Antibody reactivity was analyzed by use of a 2-D electrophoresis (2-DE) and immunoblotting technique with normal human dermal fibroblasts as previously reported [2]. When cells were confluent, cellular monolayers were first washed with phosphate buffered saline (Gibco BRL Invitrogen) containing 0.02% EDTA (Sigma-Aldrich; St. Louis, MO, USA). Fibroblasts, 106/mL, were then suspended in a sample solution extraction kit (BioRad; Hercules, CA, USA) to which 64 mM (final concentration) dithiothreitol (Sigma) was added. Cell samples were sonicated, and the supernatant was collected after ultracentrifugation (Ultracentrifuge Optima L90K, Beckman Coulter; Fullerton, CA, USA) at 150,000 g for 25 min at 4°C. Protein quantification involved the Bradford method [3].

Two-DE, 2-D blots and protein identification by mass spectrometry on 2-DE gels were as described [2, 4, 5].

For two-dimensional gel electrophoresis (2-DE), pH range from 3 to 10 for the isoelectrofocalization (IEF) and an acrylamide gradient from 7 to 18% were used, allowing tostudy a range of fibroblast antigens from 6,000 to 200,000. All instrumentation for two-dimensional gel electrophoresis (2-DE) were purchased from BioRad unless otherwise indicated. Proteins (100 µg) were isoelectrofocused using pH range linear IPG strips on the Protean IEF Cell Isoelectric Focusing System, IPG strips (18 cm) were rehydrated and focused in an automated run in the ceramic strip holders using a 9 h rehydration at 20°C followed by 12 h at 50 V, 1 h at 200 V, 1 h at 1000 V, followed by 6 h at 10,000 V and 1h at 10,000 V. Prior to the second dimension, the strips were equilibrated in a first equilibration solution then in a second equilibration solution. Equilibrated strips were sealed on the top of a 7%-18% polyacrylamide gradient gel. Gels were run initially at 40V constant for 1 h and then at 15 mA/gel.

After migration, proteins were transferred onto a polyvinylidene difluoride (PVDF) (Millipore, Bedford, MA, USA) membrane by semidry transfer (Bio-Rad) at 320mA for 90 minutes. After blocking the membranes with PBS-0.2% Tween, each membrane was incubated 4h at room temperature with one pool of sera from three patients with identical phenotype at a dilution of 1/100. Membranes were then washed before being incubated with secondary rabbit anti-human Fc-gamma antibody coupled to alkaline phosphatase (Dakocytomation, Golstrup, Denmark) for 90 min at room temperature. Immunoreactivities were revealed using the nitroblue tetrazolium–5-bromo-4-chloro-3-indolyl-phosphate substrate (Sigma). Specific reactivities and their density were determined by scanning the membranes (densitometer GS-800, BioRad). The membranes were then stained with colloidal gold (Protogold®, British Biocell International, Cardiff, UK) and subjected to a second scan to record labelled protein-spots for each membrane.

Analytical gels were stained with ammoniacal silver nitrate [6].

Images of both gels and 2D-blots were exported to Image Master 2D Platinum (version 5.0, Amersham). The number of spots was determined. A stack of the 2D-blot images before and after colloidal gold staining was acquired by the matching algorithm according to the manufacturer’s recommendations. Specific labels were then manually linked to the protein spots probed by serum IgG on both images. The algorithm automatically propagated these labels from the image of the colloidal gold-stained 2D-blot to the image of reference silver-stained.

For protein identification by mass spectrometry on 2-DE gels, 400 µg of proteins was loaded onto IPG strips. Gels were stained with 0.2% Coomassie Blue R-250 (Sigma) in 50% methanol, 10% acetic acid and destained in 50% methanol, 2% acetic acid. The relevant spots were selected by comparison of the 2D-blots with the silver-stained analytical gels and colloidal Coomassie blue-stained preparative gels. Spots of interest were excised from the gel, washed with 200 mM ammonium bicarbonate (ABC). In-gel trypsin digestion was carried out as described in a protocol based on ZipPlate (Millipore Billerica, MA, USA) with minor variations. After destaining, spots were rinsed three times with water and shrunk with 50 mM ABC/50% acetonitrile (ACN) for 20 minutes at ambient temperature. Gel pieces were then dried using 100% ACN for 15 minutes. Protein spots were then incubated in 50 mM ABC containing 10 mM DTT for 1 hour at 56°C. The solution was then replaced by 55 mM iodoacetamide in 50 mM ABC and incubated 30 minutes in the dark at ambient temperature. The protein spots were washed twice with 50 mM ABC and finally shrunk with 25 mM ABC/50% ACN for 30 minutes and dried using 100% ACN for 10 minutes. Spots were rehydrated with 20 µl of 12.5 µg/ml Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) in 40 mM ABC/10% ACN pH 8.0. Proteins were digested overnight at 37°C. After digestion, the spots were shrunk with 100% ACN and peptides were extracted with 0.2% TFA. Peptides were then desalted using C18 phase on ZipPlate. Two elutions were performed to recover products from C18 phase first with 50% ACN/0.1% TFA and then using 90% ACN/0.1% TFA. Pooled elutions were concentrated using a vacuum centrifuge and generated peptides were redissolved in 3 µl of 1% formic acid before mass spectrometry analysis.

Digested samples were spotted directly onto a 96x2 MALDI Plate (Applied Biosystems, Foster City, CA, USA) using the dried-droplet method (0.5 µl of the sample and mixed on plate with an equal volume of 5 mg/ml of alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix in ACN/water/trifluoroacetic acid (50/50/0.1%). Droplets were allowed to dry at ambient temperature. Droplets were allowed to dry at room temperature. Mass spectrometry analysis were performed on a matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF, Voyager DE-PRO mass spectrometer, Applied Biosystems) and acquired in positive ion using reflector mode. Spectra were obtained in a reflectron-delayed extraction in source over mass range of 500-3500 Dalton.

Raw spectra were treated automatically according to the Mascot wizard algorithm (MatrixScience) on NCBInr database. When non significant scoring was obtained a manual treatment was perform according to the following explanation. First, raw spectra were treated using noise filter algorithm (correlation factor 0.7) and the default advanced baseline correction. Then monoisotopic masses were generated on full detected spectrum after internal calibration using auto-digestion tryptic peptides or using external calibration using a mixture of five external standards (PepMix 1, LaserBio Labs, Sophia Antipolis, France). Mains peaks were selected according to local background and molecular weight of the protein, known peaks contamination from trypsin and keratin using PeakErazor software (Lighthouse Data, Denmark). Peptides masses were searched against a comprehensive nonredundant protein sequence database (NCBInr; version October 2006 and later) and were carried out employing four different algorithms for protein identification (Mascot (MatrixScience), ProFound (Proteometrics), MS-Fit (ProteinProspector) and Aldente (Expasy) softwares). Allowed variable modifications were oxidation of methionine, acrylamide modified cystein and carbamidomethylation of cystein. Up to one missed tryptic cleavage was considered and a mass accuracy in the range of 25 to 50 ppm was used for all tryptic mass searches. Identified proteins, defined as proteins detected with the most elevated score using at least three algorithms, are listed in Table 2 of the manuscript.

Detection and validation of Ab reactivity against Saccharomyces cerevisiae and human recombinant -enolase by ELISA

Ninety-six-well plates (MaxiSorb, Nunc, Naperville, IL, USA) were coated with 100 L purified Sc-enolase (concentration 3 g/mL) or rHu -enolase (4 g/mL) in bicarbonate buffer and incubated overnight at 4°C. Plates were then washed with PBS, and wells were saturated with 1% PBS-BSA for 1 h at room temperature. Plates were then washed 3 times with PBS. An amount of 100 L serum samples diluted (1:100) in 1% PBS-BSA was added to each well in duplicate. Mouse anti-human recombinant -enolase Ab (mouse anti-ENO1, AbNova) was used as a positive control. An amount of 1% PBS-BSA was used as a negative control. After 1 h of incubation at room temperature, plates were washed with 1% PBS-BSA and incubated with 100 L alkaline phosphatase–conjugated rabbit anti-human IgG (1:1000) (Rockland, Gilbertsville, PA) or rabbit anti-mouse IgG (1:1000) (Rockland) for 1 h. After being washed with PBS, plates were incubated with 75 L pNPP (Sigma). Optical density was monitored at 405 nm, and the readings were recorded by use of a 96-well ELISA reader (Fusion, Packard model A15360S, Rungis, France).

References

1Stupi AM, Steen VD, Owens GR, Barnes EL, Rodnan GP, Medsger TA. Pulmonary hypertension in the CREST syndrome variant of systemic sclerosis. Arthritis Rheum. 1986;29:515-24.

2Terrier B, Tamby MC, Camoin L, Guilpain P, Broussard C, Bussone G, et al. Identification of target antigens of antifibroblast antibodies in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2008;177:1128-34.

3Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54.

4Servettaz A, Guilpain P, Camoin L, Mayeux P, Broussard C, Tamby MC, et al. Identification of target antigens of antiendothelial cell antibodies in healthy individuals: A proteomic approach. Proteomics. 2008;8:1000-8.

5Guilpain P, Servettaz A, Tamby MC, Chanseaud Y, Tamas N, Garcia de la Pena-Lefebvre P, et al. A combined SDS-PAGE and proteomics approach to identify target autoantigens in healthy individuals and patients with autoimmune diseases. Ann N Y Acad Sci. 2007;1109:538-49.

6Rabilloud T. Solubilization of proteins in 2-D electrophoresis. An outline. Methods Mol Biol. 1999;112:9-19.

1