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Anal Biochem. 2015 Mar 1;472:62-6. doi: 10.1016/j.ab.2014.12.006. Epub 2014 Dec 20.

Bone protein extraction without demineralization using principles from hydroxyapatite chromatography.

Author information

1
Department of Biomedical Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12182, USA.
2
Department of Biomedical Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12182, USA. Electronic address: vashid@rpi.edu.

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.

KEYWORDS:

Ammonium phosphate; Collagen I; Demineralization free; Human bone; Osteocalcin

PMID:
25535955
PMCID:
PMC4314460
DOI:
10.1016/j.ab.2014.12.006
[Indexed for MEDLINE]
Free PMC Article

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