Cleland Timothy P, Vashishth Deepak
Department of Biomedical Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12182, USA.
Department of Biomedical Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12182, USA.
Anal Biochem. 2015 Mar 1;472:62-6. doi: 10.1016/j.ab.2014.12.006. Epub 2014 Dec 20.
Historically, extraction of bone proteins has relied on the use of demineralization to better retrieve proteins from the extracellular matrix; however, demineralization can be a slow process that restricts subsequent analysis of the samples. Here, we developed a novel protein extraction method that does not use demineralization but instead uses a methodology from hydroxyapatite chromatography where high concentrations of ammonium phosphate and ammonium bicarbonate are used to extract bone proteins. We report that this method has a higher yield than those with previously published small-scale extant bone extractions, with and without demineralization. Furthermore, after digestion with trypsin and subsequent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis, we were able to detect several extracellular matrix and vascular proteins in addition to collagen I and osteocalcin. Our new method has the potential to isolate proteins within a short period (4h) and provide information about bone proteins that may be lost during demineralization or with the use of denaturing agents.
从历史上看，骨蛋白的提取依赖于脱矿质处理，以便更好地从细胞外基质中获取蛋白质；然而，脱矿质处理可能是一个缓慢的过程，会限制对样品的后续分析。在此，我们开发了一种新型蛋白质提取方法，该方法不使用脱矿质处理，而是采用羟基磷灰石色谱法中的一种方法，即使用高浓度的磷酸铵和碳酸氢铵来提取骨蛋白。我们报告称，与之前发表的有或没有脱矿质处理的小规模现存骨提取方法相比，该方法具有更高的产量。此外，在用胰蛋白酶消化并随后进行高效液相色谱 - 串联质谱（HPLC-MS/MS）分析后，除了I型胶原蛋白和骨钙素外，我们还能够检测到几种细胞外基质蛋白和血管蛋白。我们的新方法有可能在短时间内（4小时）分离蛋白质，并提供有关在脱矿质处理过程中或使用变性剂时可能会丢失的骨蛋白的信息。
