Mitochondrial proteomic characterization of human normal articular chondrocytes.
OBJECTIVE
Mitochondrial dysfunctions have been associated with apoptosis, aging and osteoarthritis (OA). Chondrocyte mitochondrial proteins are attractive targets for the study of the metabolism of cartilage degradation. The copurification of "contaminating" proteins has been the major problem in all phases of mitochondrial proteome research. Therefore, we set up a procedure for the proteomic analysis of human chondrocyte mitochondrial proteins.


METHODS
Four types of protein extracts were obtained from primary cultured chondrocytes isolated from healthy donors: (1) initial total chondrocyte extract (CE), (2) cytosol-enriched supernatant fraction (CY), (3) crude mitochondria fraction (CM), and (4) pure mitochondria fraction (PM). Mitochondria were purified by density gradient ultracentrifugation. Mitochondrial proteins were separated by means of two-dimensional gel electrophoresis (2-DE) and silver stained. Protein spots were then identified by mass spectrometry using MALDI-TOF/TOF technology.


RESULTS
The best 2-DE reference map of mitochondrial proteome was constructed employing PM fraction. Thirty-nine percent of the identified proteins were functionally distributed in the mitochondria, 14% in the endoplasmic reticulum and 36% in the cytoplasm. Examining their biological function, 22% are involved in protein targeting, 12% in signaling, 12% in glycolysis, 10% in RNA, DNA or protein synthesis, 10% in oxidative phosphorylation and 4% in redox. The analysis of mitochondrial Mn-superoxide dismutase (SODM) revealed an age-dependent decrease of this protein.


CONCLUSIONS
PM fraction allowed the obtention of a high quality proteomic map for the study of mitochondrial proteins in human articular chondrocytes. This proteomic approach may be also efficient to analyze both quantitative and qualitative modulations of the mitochondrial proteome in human chondrocytes during aging and pathological conditions such as OA.
Ruiz-Romero
C
C
Osteoarticular and Aging Research Unit, Rheumatology Division, C.H. Universitario Juan Canalejo, Xubias 84 15006-A Coruña, Spain.
López-Armada
M J
MJ
Blanco
F J
FJ
D000293:Adolescent; D000328:Adult; D000368:Aged; D002358:Cartilage, Articular / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000737:chemistry* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008875:Middle Aged; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D013482:Superoxide Dismutase / Q000032:analysis
D024101:Mitochondrial Proteins; D020543:Proteome; D013482:Superoxide Dismutase