我们用iTRAQ方法确定了76个OA病人与对照组关节软骨有差异的蛋白。其中，LECT2 (leukocyte cellderived chemotaxin-2), BAALC (brain and acute leukemia, cytoplasmic), and PRDX6(peroxiredoxin-6) 这三个是新的OA生物标记。
1）Patients and articular cartilage
Control samples were obtained from 6 patients with femoral neck fracture (4 male and 2 female patients). The samples were collected from femoral head cartilage during bipolar femoral head replacement at Fujita Health University Hospital. OA cartilage samples were obtained from 17 patients with OA (5 male and 12 female patients). These samples were collected from the femoral heads, femoral condyles, and tibial plateaus of patients with primary OA, who underwent total hip or knee replacement surgery at Fujita
Health University Hospital (Table 2).
Samples were snap frozen at ?80°C and cut into 10?m sections by using a cryo-microtome (CM1900; Leica, Grove, IL). The sectioned samples were homogenized by sonication (T10 basic Disperser/Homogenizer; IKA) in RIPA buffer (10mL of 50mM Tris-HCl, pH 8.0, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS/g cartilage). Then, the samples were centrifuged at 20000×g at 4°C for 10min, and the supernatant was used for analysis. Amicon Ultra Centrifuge Filters 10K (Millipore, Billerica, MA) were used to concentrate proteins by centrifugation at 5000×g. Concentrated samples were stored at ?80°C until use. A random peptide column (Proteominer; BIO-RAD, Hercules, CA) was used to prepare samples for iTRAQ. High-abundance proteins in these samples were excluded, while rare proteins were collected. Eluted samples were concentrated by Amicon Ultra Centrifuge Filters 10K by centrifugation at 14000×g.
Daiki Ikeda, Hiroshi Ageta, Kunihiro Tsuchida, et al.(2013). iTRAQ-based proteomics reveals novel biomarkers of osteoarthritis. Biomarkers 18(7): 565-572.