Research Profile: A new method to extract protein from bone

from bone Elucidating the bone proteome is essential for understanding normal bone physiology as well as diseases such as osteoporosis. However, the best way to extract the necessary proteins for proteomic analysis has been a bone of contention among researchers. Because bone is mineralized and almost solid, classical protein extraction methods cannot be applied efficiently. In this issue of JPR (pp 2287–2294), Hanfa Zou, Lei Cui, and co-workers at the Dalian Institute of Chemical Physics, the Shanghai Tissue Engineering Research and Development Center, and Suzhou University (all in China) introduce an efficient method to extract proteins from bone for proteomic analysis. Most bone proteomic studies have used in vitro systems such as cultured osteoblasts and osteoclasts. However, these systems are unlikely to recapitulate the in vivo protein expression of bone cells, which are normally embedded in a hydroxyapatite matrix. Other researchers have attempted to extract proteins from bone by finely grinding it, a very labor-intensive process. Furthermore, bone pulverization tends to extract large amounts of highly abundant structural proteins such as collagens and proteoglycans, which can overwhelm the detection of lowerabundance proteins. Zou and co-workers extracted proteins from dog skull by demineralizing bone fragments in 1.2 M hydrochloric acid. Bone proteins were extracted sequentially by using three different lysis buffers for a total of four bone extracts (including the acidic extract from the demineralization). The demineralization process removes calcium and other minerals from the bone tissue and dissolves the mineral matrix while keeping most of the organic matrix intact. According to Zou, “The removal of the mineral matrix exposes the cells in the bone, which facilitates extraction of proteins. In contrast to the pulverization of bone, our approach is very simple and allows high-efficiency protein extraction.” Compared with bone pulverization, the demineralization protocol extracted ~1.3× more total protein. The researchers used a shotgun proteomics approach to identify the proteins extracted from bone. The proteins were digested with trypsin, and the resulting peptides were separated by 2D LC. Then, MS/MS analysis was used to identify the peptides and their corresponding proteins. The researchers identified 2479 unique proteins from the four bone extracts. Of these, 816 unique proteins were assigned with high confidence after the identification of at least two peptides. Using this more stringent identification criterion, the researchers reported a false-positive rate of 0.48%. “This is the first largescale proteomic analysis of bone tissue using the shotgun approach,” says Zou. “The four-step sequential protein extraction protocol is able to exhaustively