Osteoarthritis and Cartilage Vol. 15, Supplement C C213 Abstract 388 – Table 1 Protein ID Ratio T-test Name Mitochondrial proteins increased in OA THIM 1.61 0.0015 3-ketoacyl-CoA-thiolase KAD4 1.52 0.014 Adenylate kinase isoenzyme 4, mitochondrial IMMT 1.50 0.038 Mitochondrial inner membrane protein (mitofilin), isoform 1 TRAP1 1.47 0.041 TNF receptor-associated protein 1 (Hsp75) SCOT 1.41 0.035 Succinyl-CoA 3-ketoacid coenzyme A transferase 1 NDUV1 1.36 0.029 NADH dehydrogenase (ubiquinone) flavoprotein 1, mitochondrial prec. NDUS8 1.32 0.031 NADH dehydrogenase (ubiquinone) iron-sulfur prot 8, mit. precursor Mitochondrial Proteins decreased in OA OPA1 -1.34 0.0038 Dynamin-like 120 kDa protein, mitochondrial VDAC2 -1.36 0.049 Voltage-dependent anion-selective channel protein 2 HIBCH -1.36 0.049 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial precursor ECHA -1.4 0.036 Trifunctional enzyme subunit alpha, mitochondrial IMMT -1.4 0.0066 Mitochondrial inner membrane protein (mitofilin), isoform 2 ODPA -1.5 0.017 Pyruvate dehydrogenase E1 component α sub, mitoch. precursor SODM -1.5 0.011 Mn Superoxide dismutase IDHP -1.5 0.0074 Isocitrate dehydrogenase (NADP), mitochondrial precursor DHSA -1.56 0.007 Succinate dehydrogenase [ubiquinone] flavoprotein subunit GLSK -1.6 0.007 Glutaminase kidney isoform, mitochondrial precursor SODM -1.6 0.0024 Mn Superoxide dismutase IDH3A -1.69 0.015 Isocitrate dehydrogenase [NAD] subunit alpha IMMT -1.7 0.021 Mitochondrial inner membrane protein (mitofilin), isoform 3 ETFA -1.7 0.038 Electron transfer flavoprotein subunit α, mitochondrial precursor IDH3A -1.9 0.038 Isocitrate dehydrogenase [NAD] subunit alpha the pattern of normal chondrocytes (ratio OA:N≥1.3, p<0.05), including the TNFα-receptor-associated protein (TRAP1) and two subunits of the mitochondrial respiratory chain complex I. On the other hand, 60 protein spots were decreased in OA chondrocytes, including two mitofilin and two superoxide dismu- tase isoforms. Identified proteins whose cellular localization was previously described as mitochondrial are listed in the Table. Unsupervised Principal Component Analysis and hierarchical clustering allowed the recognition of OA and control samples by their protein profiles. Validation of the results was performed for the TNFα-receptor associated protein TRAP1 by immunoflu- orescence, real-time PCR and western blot assays. This protein was found to be increased specifically the deep layer of OA chondrocytes. Conclusions: This study describes the differences between the mitochondrial protein profiles of normal and OA chondrocytes. pointing out the mitochondrial alterations that take place in carti- lage degradation during osteoarthritis. 389 CYTOSKELETAL DISTORTIONS IN HUMAN OSTEOARTHRITIC CHONDROCYTES REVEALED BY A DIFFERENTIAL PROTEOME ANALYSIS S. Lambrecht 1 , G. Verbruggen 2 , P. Verdonk 2 , D. Elewaut 2 , D. Deforce 1 1 Ghent University, Gent, Belgium; 2 Ghent University Hospital, Gent, Belgium Purpose: A proteome analysis of human articular chondrocytes was conducted to identify proteins, differentially expressed in chondrocytes during the progression of osteoarthritis (OA). Methods: The proteins of 20 samples of human chondrocytes obtained from the cartilage of human knees (6 from healthy cartilage (NoNo), 7 from visually intact zones (NoOA) and 7 from visually damaged zones (OAOA) of OA-cartilage from the same knee joint) were sequentially extracted and subjected to two-dimensional gel electrophoresis. Protein expression patterns were subjected to statistical analysis and protein spots of in- terest were identified by electrospray ionization tandem mass spectrometry. Results: The chondrocytes isolated from visually intact zones of OA-cartilage showed a different expression pattern for a substan- tial number of proteins compared to chondrocytes isolated from healthy controls, hereby confirming the hypothesis that chondro- cytes undergo phenotypic alterations even before the develop- ment of visible cartilage damage. Furthermore, new players were identified in processes known to be involved in the pathogenesis of OA such as apoptosis, oxidative stress and metabolic activa- tion. Of special interest was the detection of cleaved vimentin, which was upregulated in OAOA samples. The higher abundance of vimentin cleavage products was confirmed by Western blot. The possible impact of vimentin cleavage on the chondrocyte’s cytoskeleton was illustrated by confocal microscopy analysis, which revealed a distorted vimentin organisation in OA chondro- cytes. In contrast, F-actin staining did not reveal differences. Conclusions: All together, this study revealed several differen- tially expressed proteins in osteoarthritic chondrocytes. Further validation experiments clearly point to substantial alterations in the vimentin cytoskeleton in OA-affected human articular chon- drocytes. 390 EXPLORATION OF THE PROTEOME OF HUMAN ARTICULAR CHONDROCYTES BY A SHOTGUN PROTEOMICS APPROACH S. Lambrecht 1 , M. Dhaenens 1 , G. Verbruggen 2 , D. Elewaut 2 , D. Deforce 1 1 Ghent University, Gent, Belgium; 2 Ghent University Hospital, Gent, Belgium Purpose: In recent years, some studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chon- drocyte proteome remains limited. Especially the membrane and low-abundance proteome remain largely unknown. This study aims to dig further in the proteome expressed by human knee chondrocytes, thereby providing a database for future biomolec- ular research. Methods: Chondrocytes isolated from human knee cartilage samples were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydropho- bicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line 2-D nanoLC system coupled to a Qq-TOF mass spectrometer. Results: Using extensive prefractionation based on the hy- drophobicity and molecular weight of the proteins, we were able to identify several membrane proteins, and other low-abundance