| Objective: To explore proteins associated with non-traumatic Osteonecrosis of the femoral head in adults by using high throughput proteomic approach.Methods: 5 necrotic bone tissue samples were harvested from the femoral head of patients with osteonercrosis of the femoral head, and 5 normal bone tissue samples were collected as the control group in the same patients. After bone tissues were demineralized by HCl solution, total bone proteins were extracted sequentially by using four different lysis buffers. Thereafter, multi-dimensional liquid chromatography-tandem mass spectrometry(MDLC-MS/MS) were applied to separate and identify the extracted proteins, and bioinformatics analysis was carried out then by the software tools such as TurboSEQUEST and GoMiner.Results: The in-depth proteome analysis of necrotic and normal bone tissue revealed 1233 and 999 high-confidence proteins respectively based on two peptides minimum, with the a false-positive rate of 3%. 189 differential expressed proteins were identified by spectral counting. Compared with the normal samples , the necrotic bone tissues showed that level of 104 proteins was upregulated 3 fold and the level of 50 proteins was downregulated at least 3 fold. Enrichment analysis in Gene Ontology categories of the differentially expressed gene found that 34 differential expressed proteins were highly associated with Osteonecrosis of the femoral head. Western blotting analysis of GPCR26 and Isoform 1 of Carbohydrate sulfotransferase 2 confirmed the results of mass spectrometry.Conclusion: The pathogenesis of the non-traumatic Osteonecrosis of the femoral head has been proved complicated and remains unknown. These identified differential expressed proteins might be promising biomarker candidates for early diagnosis of the devastating disease. The results of this study will affirmatively provide a new approach to understand the etiopathogenesis of the Osteonecrosis of the femoral head.
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