Patients
This study included fourteen AS patients who met the modified New York criteria [15] and ten with Rheumatoid Arthritis who fulfilled the American College of Rheumatology revised criteria [16]. Ethical permission was obtained (Oxfordshire REC 06/Q1606/139). Subjects gave informed consent. All AS patients were HLA-B*2705 positive by DNA typing. The AS patients’ mean age was 42 and the range from 28 to 69. Eight were male, seven received NSAIDs, four DMARDs and none anti-TNFs. The AS patients’ mean and standard deviation scores on the Bath Ankylosing Spondylitis Disease Activity Index, Bath Ankylosing Spondylitis Functional Index, and Bath Ankylosing Spondylitis Metrology Index scales were 5.8 +/- 2.6, 5.4 +/- 2.8, and 5.2 +/- 1.8 respectively. The RA patients’ mean age was 48 and range 18 to 68. Six were female, four received NSAIDs, six received DMARDs and none anti-TNF agents. The RA patients’ DAS score was 5.2 +/- 2.3. Fourteen HLA-B27- and four HLA-B27+ individuals, eight male with a mean age of 40 and range of 28 to 64, were included as healthy controls.
Monocyte isolation and characterization
Blood samples of 30 ml were collected in heparinized tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient centrifugation. CD14+ monocytes were separated using MACSTM CD14 Microbeads (Miltenyi Biotec) according to the manufacturers instructions. In order to confirm purity, aliquots of monocytes were washed twice with PBS, resuspended in 50 L of RPMI media (Sigma) + 0.02% azide and labelled for 45 minutes on ice with 1 L of FITC conjugated anti-CD14, PE conjugated anti-CD3, or FITC conjugated anti-CD19 (BD Biosciences). FACS analysis was carried out on a FACSCanto flow cytometer and analysed with FlowJo software (BD Biosciences).
Metabolic labelling and protein extraction
5 x 106 purified monocytes were starved at 37ºC in methionine-free RPMI for one hour. 13.25 MBq of [35S] Promix (GE Healthcare, UK) was then added and the cells were incubated overnight at 37ºC. The monocytes were then washed twice with PBS, and pelleted cells lysed in 200 L of cold buffer (50 mM NaOAc, 5 mM MgCl2, 0.5% NP40 + Protease mix(Roche) in 50mM Tris pH 7.2) for 30 minutes. The nuclear proteins were spun out at 14,000 rpm for 10 minutes at 4ºC and the proteins in the supernatant were then precipitated via methanol-chloroform precipitation (17).
Two-dimensional gel electrophoresis
Precipitated protein pellets were resuspended in rehydration buffer (7M urea, 2M thiourea, 20 mM DTT, 4% CHAPS and 0.2% pH 5 – 8 carrier ampholytes (Bio-Rad)). Protein concentration was determined with the RediPlate EZQ Protein Quantitation Kit (Invitrogen). 100 µg of protein was subjected to isoelectric focusing (IEF) using immobilized pH 5-8 strips (Bio-rad) in a PROTEAN IEF Cell (Bio-rad) in rapid ramping mode for 40,000 volt hours. Proteins were reduced with 20 mM DTT and alkylated with 30 mM iodoacetamide prior to separation on a CriterionXT 4-12% Bis-Tris 11 cm Precast Gel (Bio-Rad). Gels containing 35S-labeled samples were incubated in dimethyl-sulfoxide (DMSO) for one hour and then in a saturating solution of DMSO and 2,5-diphenyloxazol (PPO) for one hour to enhance scintillation. The PPO was precipitated in the gel by incubation in H2O for 30 minutes. The gels were dried and then exposed to film (Biomax XAR-Omat, Kodak). 100 g of monocyte protein was unlabeled and run on a duplicate gel which was silver stained for spot excision as described in (18).
Gel scanning and image analysis
Dried [35S] labelled gels were scanned and imported into PDQuestTMBasic 8.0 2D analysis software (Bio-Rad). Spots were detected using the spot detection wizard. A master gel containing the data from all gels was created. Matched sets were created by comparing each gel individually to all others, and parameters were set to detect spots with more than a two-fold difference in intensity. All results from the PDQuest software were then verified manually.
Protein excision, digestion and nano-LC-MS/MS analysis
Spots identified as upregulated in patient gels were matched up with spots on duplicate silver stained gels and excised. Excised gel pieces were reduced, alkylated and digested with porcine trypsin (Promega) as describedin(19). Liquid chromatography was performed on an UltimateTM (LC-Packings, Dionex, Amsterdam, The Netherlands) system equipped with a FamosTM autosampler. This and the 3D high-capacity ion trap mass spectrometer (Bruker Daltonics) were controlled using HystarTM 3.0 and EsquireControlTM 5.2 (Bruker Daltonics) software. Data were processed and Mascot compatible files created using DataAnalysisTM 3.2 software (Bruker Daltonics). Searches of the SwissProt and NCBInr databases were performed using MascotTM software (Matrixscience, London, UK). Individual MS/MS spectra for peptides with a Mascot Mowse score lower than 40 were excluded. All MS/MS spectra were inspected manually for a series of at least four continuous y or b ions.
Monocyte fractionation
Isolated monocytes were resuspended in 1 ml of CBL buffer (10 mM Hepes, 10 mM NaCl, 1 mM KH2PO4 , 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2-2H2O, 0.5 mM MgCl2-6H2O, 1 mM PMSF, 1 mM IAA + Protease mix(Roche), pH 7.4) and incubated for 10 minutes on ice. Cells were homogenized in a Dounce apparatus and isotonicity was restored by adding 100 µl of 2.5 M sucrose. The samples were vortexed and the nuclei were spun out at 1,500 rpm for 10 minutes at 4ºC. Supernatants were collected and spun in an ultracentrifuge for 30 minutes at 50,000 rpm, 4ºC to pellet the membrane fraction. Cytosolic material enriched in supernatants were lyophilized to a volume of 200 µl prior to methanol-chloroform precipitation. Membrane pellets were resuspended in 200 µl of solubilization buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris 1 mM PMSF, 1 mM IAA + Protease mix(Roche)) and incubated for 1 hour on ice with occasional vortexing. The membrane fractions were then ultracentrifuged at 55,000 rpm for 3 hours and soluble membrane proteins in the supernatant were precipitated via methanol-chloroform precipitation. The proteins were resuspended in 6M urea 100 mM Tris and subjected to reduction, alkylation and digestion with trypsin. Desalting was carried out using C18 Sep-Pak Cartridges (Waters) according to the manufacturers’ instructions.
Analysis by nano-UPLC-MSE tandem mass spectrometry and IPA analysis. Monocyte protein digests were subjected to liquid chromatography tandem mass spectrometry analysis (nano UPLC-MSE) using a Waters Nano-AcquityTM UPLC system coupled to a Waters QTOFpremierTM tandem mass spectrometer (Waters, Milford, MA, USA). Data was acquired in high-definition MSE mode and processed with ProteinLynx Global Server (PLGS version 2.2.5, Waters, Milford, MA, USA) to reconstruct MS/MS spectra by combining all masses with a similar retention time. MS/MS spectra (peaklists) were searched against the SwissProt/NCBInr databases using PLGS and Mascot (Matrixscience, London, UK) for the identification of proteins. The local “in-house” Mascot server is supported and maintained by the Computational Biology Research Group at the University of Oxford. Quantitative analysis of MS data was performed using the Waters Expression Profiling System (WEPSTM), and only changes greater than +/- 1.3 with a p-value of < 0.05 were included in the analysis. Data were analysed through the use of Ingenuity Pathway Analysis (Ingenuity® Systems, IPA identified significant signalling pathways using the Right Tailed Fischer’s Exact Test. The Benjamini-Hochberg method was used to adjust p-values for multiple comparisons.
Peptide synthesis, in vitro digestion, analysis and quantification
Extended peptides (LELRSRYWAIRTRSGSN and NQQITANRELQQELAAA) containing the HLA-B27 epitopes SRYWAIRTR (20), and NRELIQQEL (21) were synthesized by solid-phase F-moc chemistry and purified to >98% by HPLC chromatography. 1 µg of proteasome (Biomol) was incubated with 10 µg of peptide in a final volume of 100 µL of 20 mM Hepes, pH 7.8, 2 mM MgCl, 2 mM DTT with or without PA28 (1 µg) for 2 hours at 37ºC. Digests were stopped with 0.1 volume of triformic acid. All digests were repeated three times. Proteasomes were pelleted by ultracentrifugation at 55,000 rpm for 4 hours at 4ºC. 2.5 µL of supernatant containing peptide digest was then analysed using a nano UPLC-MSE mass spectrometer. Ion extraction chromatograms corresponding to the peptide masses were generated for each time point in the presence and absence of the PA28 complex.