title: Bone protein extraction without demineralization using principles from hydroxyapatite chromatography.
abstract: 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.
authors.affiliation: Department of Biomedical Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12182, USA.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Cleland
authors.affiliation: Department of Biomedical Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12182, USA. Electronic address: vashid@rpi.edu.
authors.forename: Deepak
authors.initials: D
authors.lastname: Vashishth
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002853:Chromatography, Liquid; D017886:Durapatite / Q000737:chemistry*; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D013977:Tibia / Q000737:chemistry*
chemical_list: D016326:Extracellular Matrix Proteins; D017886:Durapatite
keywords: Ammonium phosphate; Collagen I; Demineralization free; Human bone; Osteocalcin