Subjects
Ten patients with AS were recruited from an outpatient rheumatology clinic in Hanyang University Hospital for Rheumatic Diseases, Seoul, South Korea. An additional 30 patients treated at Keimyung University Dongsan Hospital, Daegu, South Korea were recruited as disease control groups (10 OA, 10 gout, and 10 RA). The patients met the 1984 New York criteria for ankylosing spondylitis [17] and the American College of Rheumatology criteria for RA [16], knee OA [18], and acute gout [19]. We collected demographic and clinical data from the subjects, including age, gender, disease duration, blood chemistry, and concomitant treatment. Table 1 shows the diagnosis, clinical data, and current treatments of each patient enrolled in this study. The samples were obtained after getting informed consent from the patients. The study was approved by the ethical committee of the Keimyung University Dongsan Hospital (IRB 2015-12-022).
Synovial fluid sample collection and processing
Synovial fluid was collected by arthroplasty in patients with knee joint pain and swelling. Contaminated bloods during arthroplasty and samples where synovial fluids were generated for reasons other than the respective diseases were excluded. After centrifugation at 500 x g for 10 min, five 1 mL vials of supernatant and 1 vial of sediment from each sample were stored at -80 °C through the Keimyung University Dongsan Hospital Human Resource Bank.
Immunodepletion of abundant proteins with MARS cartridge
We used a MARS spin cartridge (Agilent; Santa Clara, CA, USA) to remove highly abundant proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin), which occupy about 70% of the total SF proteome. The 0.22 μm membrane filter (Agilent; Santa Clara, CA, USA) was used to remove particulates from the fluid samples by centrifugation at 100 x g for 1.5 min. The flow-through was mixed with Buffer A LOAD/WASH (Agilent; Santa Clara, CA, USA) and depleted according to the manufacturer’s instructions (Agilent Technologies, USA). During the depletion, flow-through was collected, and protein concentration was determined using a BCA assay. The column was routinely regenerated by eluting bound high-abundance proteins with buffer B and neutralizing with buffer A before further use. The acquired proteins were directly digested for total proteomic analysis.
Peptide sample preparation
In-solution tryptic digestion and peptide cleanup were simultaneously performed in a 96-well plate for high reproducibility. Each depleted sample was supplemented with 8 M Urea in 100 mM ammonium bicarbonate (Sigma, St. Louis, MO, USA) and incubated for 20 min at room temperature. The samples were homogenized by vortexing and sonication thrice. To each sample, DTT was added to be 10 mM for protein reduction at RT for 1 hr. Then, IAA was added to be 30 mM for cysteine bond alkylation in the dark at RT for 30 min. Samples were then diluted with 100 mM ABC prior to the addition of trypsin at a 1:50 trypsin: sample ratio, and incubated at 37℃ overnight. The trypsin was inactivated by acidification with 0.4% TFA. The acidified digests were immediately processed using a Sep-Pak C18 96-well plate (100 mg C18 sorbent per well, Waters). The peptides were eluted with 80% acetonitrile (ACN) and then dried in a vacuum centrifuge.
LC-MS/MS experiments
We performed a comprehensive label free quantitative proteomics analysis using LC-MS/MS to identify proteins in synovial fluid. LC−MS/MS analysis of 40 peptide samples was carried out on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to a nanoACQUITY UPLC (Waters, Milford, MA) with an in-house-packed trap (150 μm i.d. × 3 cm) and analytical column (75 μm i.d. × 100 cm) using 3 μm Jupiter C18 particles (Phenomenex). The LC gradient was as follows: from 5% to 40% solvent B (acetonitrile with 0.1% formic acid) for 130 min, then 40% to 80% solvent B for 5 min, holding at 80% solvent B for 10 min, and then equilibrating at 95% solvent A (water with 0.1% formic acid) for 30 min. Full MS data were acquired in a scan range of 375–1,575 Th at a resolution of 60,000 at m/z 200, with an automated gain control (AGC) target value of 4.0×105 and a maximum ion injection time of 50 ms. The maximal ion injection time for MS/MS was 50 ms at a resolution of 15,000 and an AGC target value of 5 x 104. The dynamic exclusion time was set to 30 s. The resulting 40 .raw files were analyzed using MaxLFQ in Maxquant software (Figure 1).
Data analysis
MaxQuant (v. 1.5.1.2) [20] was used to compare acquired spectra to the Uniprot human database (obtained in June 2018). Carbamidomethylation of cysteine was selected as a fixed modification and N-acetylation and oxidation of methionine were set as variable modifications. A false discovery rate (FDR) cutoff of 1% was applied at the peptide spectrum match (PSM) and protein levels. An initial precursor mass deviation of up to 4.5 ppm and a fragment mass deviation of up to 20 ppm were allowed. Protein identification required at least one peptide using the ‘razor plus unique peptides’ setting. Proteins were quantified using the XIC-based label-free quantification (LFQ) algorithm in MaxQuant [21]. The ‘match between runs’ option was used for nonlinear retention time alignment. The match time window was 0.7 min, and the alignment time window was 20 min. Further statistical and bioinformatics analysis was performed using Perseus software (v. 1.5.3.2). Proteins representing hits to the reverse database, contaminants, and proteins only identified by sites were eliminated prior to analysis. A minimum of three valid values across each clinical group was required for each protein group for quantification. The LFQ intensity for each protein group was compared between clinical groups following log2 transformation. Statistical analysis of the log2-transformed data was performed using two samples t-test (p value < 0.05). Proteins with ≥ 1.5 fold change between AS and other patient groups were considered to be differentially expressed proteins (DEPs) for AS group. The resulting DEPs were submitted to gene ontology annotation enrichment analysis.
Enrichment and network analysis
To explore functional enrichment in the identified proteins in each of the clinical groups, GO analysis using DAVID (version 6.8) [22] was performed. We identified biological processes and KEGG pathways [23] that were enriched in our dataset with p values less than 0.05 [22]. To construct a network describing the enriched GO terms, we selected DEPs that are involved in enriched cellular processes. We then built a protein network model using protein interaction information obtained from STRING version 11 database [24]. The interaction network models were visualized using Cytoscape [25].
Western blot verification
We verified the results of our LC-MS/MS analysis using western blotting to confirm the abundance of selected proteins in the SF of patients from each of the clinical groups. SF was clarified by incubation with hyaluronidase (Sigma, H3884) at room temperature for 10 min, and then diluted 1:10 in RIPA buffer (ThermoFisher, Waltham, MA) containing protease inhibitor (Roche Diagnostics GmbH, Mannheim) and phosphatase inhibitor (Roche Diagnostics GmbH, Mannheim, MA). The diluted sample was incubated on ice for five minutes, then transferred into new tubes and centrifuged at 10000 x g at 4°C for 15 min. Equal amounts of protein (30 ug) in each sample were aliquoted following protein quantitation by BCA assay (BCA Protein Assay, ThermoFisher, Waltham, MA). Samples were mixed with sodium dodecyl sulfate (SDS) loading buffer and separated using SDS polyacrylamide gel electrophoresis (Western Blotting Kit, Hoefer Inc., Holliston, MA). Proteins were then transferred onto nitrocellulose membranes (Amersham, Chicago, IL, USA), blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and immunoblotted with the appropriate primary and secondary antibodies. The antibodies used were as follows: rabbit polyclonal antibody for C9 (1:5000, PA5-29093, ThermoFisher, Waltham, MA), CFHR5 (1:500, ab175254, Abcam, Cambridge), MMP3 (1:5000, ab52915, Abcam, Cambridge, MA), Peroxidase-conjugated AffiniPure Donkey Anti-Rabbit IgG (H+L) (1:10,000, Jackson Immunoresearch, Baltimore Pike, West Grove, PA).