Differential proteomic screening and identification for non-traumatic necrotic femoral osseous tissue

Abstract

Currently, there is a lack of effective early screening and detection methods for femoral head necrosis. Current research on most orthopedic diseases focuses on proteomics in the preliminary stage. The recent fluorescence differential in gel electrophoresis (DIGE) has advantages such as a high reproducibility, high sensitivity, high throughput, and high dynamic range. It is currently one of the most widely used quantitative proteomic research means. We conducted this study to investigate the pathogenesis of non-traumatic femoral head necrosis using the fluorescence DIGE to screen non-traumatic femoral head necrosis based on proteomics and provide a theoretical basis for screening possible biomarkers and molecular targeted treatment. The DIGE technique was used to separate the protein. An electrophoretogram was established on the basis of scanning and analysis. Identification and a bioinformatics analysis were conducted for the differential protein. The protein with differential expression of over 2-fold was excavated and ionized by means of substrate assisted laser desorption. The flight time was identified with a mass spectrometer (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF/TOF). The formation on sequences, structures and functions of these proteins were obtained through database retrieval. Western blot analysis was used to verify the differential protein expression and the reliability of the DIGE result was verified. DIGE was used to successfully separate 1,500±40 protein spots. There were 252 significant differential protein spots. The Ettan™ Spot Picker automatic work station was used to excavate 49 significant differential protein spots with expression difference over 2-fold. The MALDI-TOF/TOF mass spectrometer was used to identify these differential protein spots. Six proteins were identified in total, which include apolipoprotein A1 (APOA1), fibrous protein original chain, fibrous protein original chain, serum albumin, sulfur-oxygen protein peroxiredoxin 2 (PRDX2) and actin. APOA1 and PRDX2 were subject to western blot analysis detection; results were consistent with the DIGE result. Based on an analysis of the biological information, these proteins may be associated with the incidence and progression of femoral head necrosis.

Keywords: bioinformatics; femoral head necrosis; fluorescence differential gel electrophoresis; mass spectrum; proteomics.

Figure 1.

Mass spectrum identification Mowse score of differential protein. (A) Mowse scores of apolipoprotein A1 (APOA1), (B) peroxiredoxin 2 (PRDX2), (C) ACTG1, (D) ALB, and (E and F) FGB proteins. The score of a peptide fragment of APOA1 was 314; the score of PRDX2 was 85; ACTG had 2 adjacent peptide fragments with ALB of 480, FGB of 200, and FGG of 198. Appropriate protein types were obtained by a mass spectrometric analysis and a database comparison.

Figure 2.

Protein expression levels of apolipoprotein A1 (APOA1) and peroxiredoxin 2 (PRDX2) in the necrosis group and the control group. (A) Protein expression levels of APOA1 in the control and necrosis groups (western blot analysis, P<0.05). (B) Protein expression levels of PRDX2 in the control and necrosis groups (western blot analysis, P<0.05) (internal reference: actin).