Bone protein extraction without demineralization using principles from hydroxyapatite chromatography

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 (4 h) and provide information about bone proteins that may be lost during demineralization or with the use of denaturing agents.