Jiang Xiaogang, Ye Mingliang, Jiang Xinning, Liu Guangpeng, Feng Shun, Cui Lei, Zou Hanfa
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
J Proteome Res. 2007 Jun;6(6):2287-94. doi: 10.1021/pr070056t. Epub 2007 May 8.
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.
探索骨蛋白质组对于理解骨组织生理/病理过程的机制而言是一项重要且具有挑战性的任务。然而，用于软组织和细胞的经典蛋白质提取方法并不适用于骨组织。因此，开发高效的蛋白质提取方法对于骨蛋白质组分析至关重要。我们在本研究中发现，当骨组织用盐酸（HCl）脱矿质时，蛋白质提取效率显著提高。我们开发了一种用于骨组织大规模蛋白质组分析的顺序蛋白质提取方法。首先用HCl溶液对骨组织进行脱矿质处理，然后使用三种不同的裂解缓冲液进行提取。由于HCl溶液中也存在大量酸溶性蛋白质，除了收集提取的裂解缓冲液中的蛋白质外，还收集脱矿质HCl溶液中的蛋白质用于蛋白质组分析。对收集的蛋白质组分进行自动二维液相色谱-串联质谱分析，结果鉴定出6202个独特肽段，这些肽段匹配2479个独特蛋白质。所鉴定的蛋白质在蛋白质种类和功能上具有广泛的多样性。本研究中观察到了40多种先前报道的骨特异性蛋白质和15种潜在的蛋白质生物标志物。结果表明，所开发的骨组织蛋白质提取方法，也是首次对骨进行的大规模蛋白质组学研究，对于骨蛋白质组的全面分析非常有效，可能有助于阐明骨疾病的机制。
