Bone protein extraction without demineralization using principles from hydroxyapatite chromatography

Timothy P. Cleland, Deepak Vashishth

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

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

Original languageEnglish
Pages (from-to)62-66
Number of pages5
JournalAnalytical Biochemistry
Volume472
DOIs
StatePublished - 1 Mar 2015
Externally publishedYes

Keywords

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

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