Participants
Synovial tissue samples were obtained from female patients who suffered from knee OA or knee injury and underwent surgical therapy from Nov. of 2018 to April of 2019 in Sichuan Provincial People’s Hospital. To be specific, patients who fitted the following criterion were included: (1) female patients with knee OA or knee injury, (2) aged at 20~70y, (3) without other maladies of bone and articular, or any pre-therapy history of the knee OA or knee injury, (4) signed the study consent and was willing to be follow-up. Although 21 patients were included, only samples from 8 patients were applied for proteomic analysis regarding their comparability.
Detection of blood plasma hormone levels
The peripheral blood used for estrogen testing was obtained on the morning of the operative day by nurses and was sent to the Hospital’s testing center immediately.
Protein preparation and digestion
The synovial tissue (ST) samples were obtained during the surgery and immediately stored in liquid nitrogen. Protein extraction was performed according to the requirements of MS analysis. Briefly, 20mg of ST sample was ground and sonicated (Q800R sonicator, QSONICA, Newton, Connecticut, USA) in 1ml lysis buffer (7M urea, 4% SDS, 1mM phenylmethane sulfonyl fluoride (PMSF), 2mM EDTA, 10mM dithiothreitol (DTT), 1×protease inhibitor cocktail (Sigma-Aldrich, St. Louis, USA) in 30mM HEPES, pH 7.4). The insoluble content in the lysate was discarded by centrifugation at 13000g and 4 °C for 10min. The protein concentration of the supernatant was determined with a BCA Protein Assay Kit (Thermo Fisher Scientific, Bonn, Germany) and the quality of the samples was evaluated via the SDS-PAGE method. The samples were then precipitated with 15% trichloroacetic acid for 2 h at 4 °C and washed with acetone. The precipitates were redissolved in triethylammonium bicarbonate buffer. The solutions were then digested with trypsin (Promega, Madison, WI, USA) at an enzyme/ substrate ratio of 1:50 at 37 °C for 16h.
TMT labeling, HPLC fractionation
The digested peptide mixtures were first desalted using a Sep-Pak C18 column (100mg, 1cm3; Waters Corporation, Milford, MA, USA). TMT labeling was performed according to the manufacturer’s instructions. Briefly, anhydrous acetonitrile was mixed with TMT Reagents pre-equilibrated to room temperature. The mixture was added to each sample, and the purified samples were chemically labeled with the TMT10plex™ Isobaric Label Reagent Set (Thermo Scientific, Rockford, CA, USA) for 2h at RT. The samples were then pooled and dried by vacuum centrifugation. The dried samples were acidified with 0.1% trifluoroacetic acid and desalted using Sep-Pak C18 (100mg, 1cm3 ; Waters Corporation, Milford, MA, USA) and fractionated through a BEH C18 column (2.1×150mm, 1.7μm, 130Å) using ACQUITY UPLC® H-Class System (Waters Corporation, Eschborn, Germany) at a flow rate of 250μl/min.
LC-MS/MS analysis
The eluted fractions were collected and combined into 12 fractions for further LC-MS/MS analysis. The settings of MS were as follows: ESI voltage, 2kV; inlet capillary temperature, 300°C; full-scan automatic gain control (AGC) target, 5×105 ions at 60,000 resolution; scan range, 400–1600m/z; Orbitrap full-scan maximum injection time, 50ms; MS/MS scan AGC target, 2×105 ions at 30,000 resolution; and maximum rejection, 150ms.
Evaluation of clinical outcomes
Just the day before and one month after the surgery, joint functions of patients were evaluated by scores of Visual Analogue Score (VAS), Lysholm, The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). All the evaluations were conducted by the same group of doctors.
The analysis of reactions and pathways among proteins
The immune or endocrine system-related proteins which were insignificant abundances between old OA patients and young OA patients were selected out by the ClueGO tool of Cytoscape (v3.7.2) and were analyzed with Reactions and Pathways of Reactome program provided online (https://reactome.org/)
Statistical analysis
The proteomic raw data was initially processed with Proteome Discovery 1.4 (Thermo Fisher Scientific, Waltham, MA, USA). KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/) [12], a web server for gene/protein functional annotation and functional gene set enrichment, was used for pathway and Gene Ontology functional category enrichment analysis. The false discovery rate (FDR) was calculated by the method described by Benjamin. Significant results were determined based on FDR-adjusted p-value ≤ 0.05. SPSS version 17.0 (SPSS Inc., Chicago, IL) was used for the data analysis, and P value < 0.05 was considered as statistically significant. The data of BMI, Mean age, VAS, Lysholm, WOMAC scores were shown as the mean ± standard deviation. The student's t-test was applied for comparing the means of two independent groups and one-way ANOVA analysis was used to compare the means among three groups. Pearson's correlation and linear regression analysis were used to evaluate the correlation between the concentration of estrogen and the VAS, Lysholm as well as WOMAC scores.