Sample collection and processing
The samples were collected after obtaining informed consent of the patients and approval from the Institutional Ethical Committees of the Armed Forces Medical College, Pune, Fortis Hospitals, Bangalore and Command Air Force Hospital, Bangalore. Synovial fluid samples were collected from the affected joints of 10 OA patients, clinically diagnosed as per the criteria of American College of Rheumatology. These 10 OA patients included 7 females and 3 males with an average age of 65 years. Approximately 5 ml of synovial fluid was aspirated from each patient in heparin containing BD vacutainers (Becton, Dickinson and Company, New Jersey). The synovial fluid was then centrifuged at 1,500 g for 15 minutes and the supernatants were then filtered by using 0.22 μm filters (Catalog number: SLGV033RS Millipore, Massachusetts, USA) and stored at −80˚C until further processing. Twelve mg of protein isolated from five OA synovial fluid samples was pooled and depleted using Human 6-Multiple Affinity Removal LC Column (MARS-6) (Agilent Technologies, Santa Clara, USA) as per manufacturer’s instructions. The six most abundant proteins that are depleted using Human MARS-6 column are albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1 antitrypsin. For each round of depletion, 1 mg protein was loaded onto the column and 12 such depletion runs were carried out. The elution of proteins was monitored at 280 nm. The depleted synovial fluid samples from each round were pooled and their protein concentration was estimated by Lowry’s method
. Protein from the depleted and pooled protein sample was subsequently fractionated by SDS-PAGE at protein level and by, strong cation exchange (SCX) chromatography and pI-based OFFGEL electrophoresis at peptide level.
SDS-PAGE and in-gel digestion
300 μg of OA synovial fluid protein depleted of abundant proteins was resolved on a 10% SDS-PAGE (16X18cm). The gel was then stained using colloidal Coomassie blue. Twenty eight gel bands were excised and destained using 40 mM ammonium bicarbonate in 40% acetonitrile (ACN). In-gel digestion was carried out as described previously
. The sample was subjected to reduction using 5 mM DTT (60˚C for 45 minutes) followed by alkylation using 20 mM iodoacetamide (room temperature for 10 min in dark). Trypsin digestion was carried out at 37˚C for 12–16 hrs (Catalog number: V5111 Sequencing grade, Promega, Madison, WI, US). Peptides were extracted from gel pieces sequentially using 0.4% formic acid in 3% ACN twice, once using 0.4% formic acid in 50% ACN and once using 100% ACN. The extracted peptides were dried and stored at −80˚C until LC-MS/MS analysis.
Five hundred μg of depleted synovial fluid protein was reconstituted in 40 mM ammonium bicarbonate. It was then reduced (5 mM DTT), alkylated (20 mM iodoacetamide) and digested overnight using trypsin as mentioned above.
Strong cation exchange (SCX) chromatography
SCX was carried out as described earlier
. Briefly, 200 μg of digested peptide mixture was acidified using 1 M phosphoric acid and equilibrated with 10 mM potassium phosphate buffer containing 25% acetonitrile, pH 2.85 (solvent A) and fractionated using SCX on a Polysulfoethyl A column (PolyLC, Columbia, MD) (300 Å, 5 μm, 100 × 2.1 mm) using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, USA) containing a binary pump, UV detector and a fraction collector. The peptides were eluted using a salt gradient (0 to 100%) between solvent A and solvent B (10 mM potassium phosphate buffer containing 25% acetonitrile, 350 mM KCl, pH 2.85). Twenty six fractions obtained from the fractionation were completely dried, reconstituted in 0.1% trifluoroacetic acid, and further desalted using stage-tips packed with C18 material
. Desalted fractions were dried in speedvac and reconstituted in 10 μl of 0.1% TFA prior to reversed-phase (RP) liquid chromatography based tandem mass spectrometry (LC-MS/MS) analysis.
Approximately 300 μg of in-solution digested depleted tryptic peptides was used for isoelectric point based fractionation using Agilent’s 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, USA). As per the manufacturer’s protocol, peptides were separated using pH 3–10 IPG strip. The peptides were focused for 50kVh with maximum current of 50 μA and maximum voltage set to 4000 V. Twelve fractions were collected after fractionation and then acidified using 1% TFA prior to sample cleaning using stage-tips
Lectin affinity enrichment
Approximately 10 mg of the total protein pooled from five OA samples was diluted in 10 mM phosphate buffer, pH 7.8. For glycoprotein enrichment, the samples were incubated with a mixture of three agarose conjugated lectins- concanavalin A (Con A), wheat germ agglutinin and jacalin (Vector labs, USA) for 12 h at 4˚C. The beads were then washed three times using wash buffer (10 mM phosphate buffer, pH 7.8) and the bound proteins were eluted using a mixture of carbohydrates (100 mM each of N-acetylglucosamine, melibiose and galactose). The eluate was dialyzed to remove free sugars and then concentrated using 3 kDa cut-off filters. The protein concentration was estimated by Lowry’s method. Two hundred and fifty μg of the enriched protein fraction was then resolved by SDS-PAGE. Twenty six gel bands were excised and subjected to in-gel trypsin digestion procedure as described in the previous section
. Two hundred and fifty μg of the enriched glycoprotein was also subjected to SCX fractionation as described earlier. Twenty fractions were collected and desalted using stage tips as mentioned above.
Tandem mass spectrometric analysis of 112 fractions obtained from depleted total proteome and enriched glycoproteome was carried out using LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Bremen, Germany) interfaced with Agilent 1200 (Agilent technologies, Santa Clara, CA, USA) nano liquid chromatography system. The LC system consisted of an enrichment column (3 cm × 75 μm, C18 material 5 μ particle size, 100 Å pore size) and an analytical column (10 cm × 75 μm, C18 material C18 material 5 μ particle size, 100 Å pore size) packed using pressure injection cell. Electrospray ionization source was fitted with an emitter tip 8 μm (New Objective, Woburn, MA) and maintained at 2000 V ion spray voltage. Peptide samples were loaded onto an enrichment column in 0.1% formic acid, 5% ACN for 15 min and peptide separation carried out using a linear gradient of 7-35% solvent B (90% ACN in 0.1% formic acid) for 60 minutes at a constant flow rate of 350 nl/min. Data was acquired using Xcalibur 2.1 (Thermo Scientific, Bremen, Germany). The MS spectra were acquired in a data-dependent manner in the m/z range of 350 to 1800 and survey scans were acquired in Orbitrap mass analyzer at a mass resolution of 60,000 at 400 m/z. The MS/MS data was acquired in Orbitrap mass analyzer at a resolution of 15,000 at 400 m/z by targeting top 20 most abundant precursor ions for fragmentation using higher energy collisional dissociation activation at 39% normalised collision energy. Single and unassigned charge state precursor ions were rejected. The dynamic exclusion option was enabled during data acquisition with exclusion duration of 60 seconds. Lock mass option was enabled for real time calibration using polycyclodimethylsiloxane (m/z, 445.12) ions
Mass spectrometry data was analyzed using multiple search engines to maximize the peptide identifications. Proteome Discoverer 1.3 (Thermo Scientific, Bremen, Germany) was used to carry out the peak list generation and database searches. Precursor mass range of 500 to 8,000 Da and signal to noise ratio of 1.5 were used as the criteria for generation of peak list files. NCBI Refseq 49 human protein database with known contaminants (32,967 entries) was used as a reference database. Sequest and Mascot algorithms were used to carry out database searches. The parameters used for database searches include trypsin as a protease with allowed one missed cleavage, carbamidomethyl cysteine as a fixed modification, and oxidation of methionine as a dynamic modification. Precursor ion mass error window of 20 ppm and fragment ion mass error window of 0.1 Da were allowed. The raw data obtained were searched against decoy database to calculate 1% false discovery rate cut-off score
. Spectra that matched to the contaminants and those that did not pass the 1% FDR threshold were not considered for analysis.
Multiple reaction monitoring (MRM)
MRM assays were developed to validate the results of LC-MS/MS analysis for three target proteins. Skyline 2.1 was used for method development, data analysis and interpretation of the MRM results
. Proteotypic peptides for each protein were selected from the discovery LC-MS/MS experiments. Preference was given to proteotypic peptides with precursor charge +2 that did not contain cysteine or methionine. A minimum of four transitions were monitored for each peptide. Equal protein amounts from the individual OA synovial fluid samples were subjected to trypsin digestion as described earlier
. MRM of each sample was carried out in triplicates on TSQ Quantum Ultra (Thermo, San Jose, CA) interfaced with Easy nanoLC II (previously Proxeon, Thermo Scientific, Bremen, Germany). Peptides were enriched on a trap column (5 μm, 75 μm × 2 cm.) for 5 minutes with solvent A (5% ACN in 0.1% formic acid). The peptides were separated on analytical column (3 μm, 75 μm × 10 cm) with a linear gradient of 7-35% solvent B (95% ACN in 0.1% formic acid) for 60 min at a constant flow rate of 300 nl/min. Both columns were packed in-house using Magic C18 AQ (Michrom Bioresources). Spray voltage of 2.5 kV was applied and ion transfer tube was maintained at 275°C. MRM data was acquired with Q1 and Q3 set at resolution of 0.4 and 0.7 respectively. The collision energy for each transition was optimized in Skyline based on the preliminary results
Determination of the relative abundance of OA synovial fluid proteins
The relative abundance of proteins in OA synovial fluid was determined by calculating normalized spectral abundance factors (NSAF) for each protein identified in the study as previously described
. NSAF for a protein k was calculated by dividing the total number of peptide spectral matches (S) identified for protein k by protein length (L) and then divided by the sum of S/L ratio for all proteins.
Gene Ontology (GO)
 analysis was done to identify the biological processes and the molecular function associated with the identified proteins. Subcellular localization, post-translational modifications, transmembrane domain and signal peptide information of the identified proteins were obtained from Human Protein Reference Database (HPRD) (http://www.hprd.org), which is a GO compliant database