Proteome analysis for the identification ofin vivo estrogen-regulated proteins in bone

Abstract

Estrogen deficiency results in a reduced bone mass, which can be prevented by treatment with estrogens. This study used a proteomic approach for the first time to obtain a global perspective of estrogens' effects on whole-bone proteins. Bone proteome profiles were examined in three groups of mice: (1) sham-operated with normal ovarian functions, (2) ovariectomised and (3) ovariectomised with estrogen replacement therapy. Bone proteins extracted from the humerus were separated by 2-DE and visualised by CBB colloidal staining. Spot detection and quantification was done by image analysis. Differentially expressed proteins were identified by MS and database search, using peptide mass fingerprint and peptide sequence analysis. Differential expression analysis in the three experimental groups showed significant changes for 14 proteins. These included proteins related to bone metabolism, cytoskeleton components and energy metabolic pathways. Our data suggest that some proteins related to cytoskeleton and to energy pathways, such as tropomyosins, aconitase 2 and enolase beta, might be new molecular targets responsive to the effects of estrogen. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the pleiotropic effects of estrogens on bone.

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

“…Proteins could also be extracted from bone tissue by grinding the undemineralized bond tissue in the lysis buffer. 10,11,18 This protocol was also applied to extract proteins in undemineralized bone in this study. The amount of protein extract was determined to be 3.31 mg protein/g bone tissue (Table 1), which was also lower than that of demineralized bone.…”

“…Therefore, it is necessary to develop efficient methods for protein extraction from bone tissue. In previous reports of bone proteome analysis, 10,11,18 the bones were first ground to powder, and the proteins were extracted by incubation of the powder in lysis buffer. However, mechanically breaking bones into powder was laborious, especially for large animal's bone.…”

“…Recently, the extraction of proteins directly from bone for proteome analysis had been reported. 10,11 The extracted proteins were first separated by 2D gel electrophoresis (2DE), the interested spots were then excised, and proteins inside were identified by mass spectrometer (MS). But the analysis of extreme proteins (extremely basic or acidic, extremely small or big, extremely hydrophobic) was still a substantial challenge for 2DE.…”

Potential biomarkers associated with diabetic glomerulopathy through proteomics

“…The MASCOT score !65 and the peptide sequence coverage !20% are considered as a potent candidate. 18 …”

“…[15][16][17] Briefly, aliquots of renal glomerular tissue extracts (150 mg) dissolved in 250 mL rehydration buffer containing 8 M urea, 2% CHAPS, 1% dithiothreitol, 0.5% IPG (pH 4-7) and bromophenol blue are loaded onto immobilineÔ strips (pH 4-7, 13 cm; GE healthcare BioSciences AB, Uppsala, Sweden). The strips are hydrated and IEF at 32,000 V/h, 20 C using an EttanÔ IPGphor II/3 isoelectric focusing unit (GE Healthcare Bio-Sciences AB).…”