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

Abstract

Exploring bone proteome is an important and challenging task for understanding the mechanisms of physiological/pathological process of bone tissue. However, classical methods of protein extraction for soft tissues and cells are not applicable for bone tissue. Therefore, method development of efficient protein extraction is critical for bone proteome analysis. We found in this study that the protein extraction efficiency was improved significantly when bone tissue was demineralized by hydrochloric acid (HCl). A sequential protein extraction method was developed for large-scale proteome analysis of bone tissue. The bone tissue was first demineralized by HCl solution and then extracted using three different lysis buffers. As large amounts of acid soluble proteins also presented in the HCl solution, besides collection of proteins in the extracted lysis buffers, the proteins in the demineralized HCl solution were also collected for proteome analysis. Automated 2D-LC-MS/MS analysis of the collected protein fractions resulted in the identification of 6202 unique peptides which matched 2479 unique proteins. The identified proteins revealed a broad diversity in the protein identity and function. More than 40 bone-specific proteins and 15 potential protein biomarkers previously reported were observed in this study. It was demonstrated that the developed extraction method of proteins in bone tissue, which was also the first large-scale proteomic study of bone, was very efficient for comprehensive analysis of bone proteome and might be helpful for clarifying the mechanisms of bone diseases.

Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing

“…However, they identified only 133 proteins, because they analyzed bone proteins using a one-step method without a demineralization stage. The other report showed only that bone proteins extracted from the skull bone of an adult beagle are carried using a demineralization step (23). There are no reports regarding the interaction between alveolar bone and soft tissue yet.…”

“…This method was based on a recently reported sequential protein extraction protocol that was used to extract proteins from skull for comprehensive analysis of its proteome. Two-dimensional high-performance liquid chromatography-tandem mass spectrometry (LC-MS/ MS) was then applied to analyze the protein extracts, enabling the identification of 2479 proteins (23). We employed a similar method to extract and identify proteins in tooth alveolar bone.…”

An activity-maintaining sequential protein extraction method for bioactive assay and proteome analysis of velvet antlers

“…Nevertheless, classical single-solvent or parallel-solvents protein extraction methods are insufficient for proteome profiling and bioactivity study of velvet antlers due to the complexity of sample. In previous reports on proteome analysis of cartilage [10] and bone [11], the sequential protein extraction method improved significantly the number of identified proteins, suggesting that more proteins might be extracted if multiple complementary extraction buffers were sequentially used. In addition, many studies have demonstrated that the extracts of velvet antlers possessed widely biological activities [12][13][14].…”

Skeletal Malformation on Balb-C Foetal Mice (Mus musculus) Administered by Immature Pineapple Fruits Extract

“…Molecular techniques can be done by conducting the skeletal proteome analysis to evaluate the teratogenic effects on the foetal skeleton. Proteome analysis gives the information about mechanisms of physiological/pathological process in bone tissues [6]. This research was performed to examine the effect of the immature pineapple fruits extract on the occurrence of skeletal malformation and identify the protein profiles of skeleton on Balb-C foetal mice.…”