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 utilizes 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 previously published small-scale extant bone extractions, with and without demineralization. Furthermore, after digestion with trypsin and subsequent 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 in a short period (4 hrs) and provide information about bone proteins that may be lost during demineralization or with the use of denaturing agents.

Influence of carboxylation on osteocalcin detection by mass spectrometry

“…This result is similar to our previous results detecting only the N-terminal peptide of OC from human samples without prior purification in the presence of a large number of collagen I peptides. [49] Removal/depletion of the collagen I or enrichment of osteocalcin by purification [37] may help to better identify this protein from bone samples by MS.…”

Bone protein “extractomics”: comparing the efficiency of bone protein extractions ofGallus gallusin tandem mass spectrometry, with an eye towards paleoproteomics

“…In either case, for studies specifically seeking NCPs in low protein samples (e.g., osteonectin in fossils) it is imperative that any demineralization fractions be analyzed rather than discarded, regardless of comparatively minute amounts of extract. Conversely, extraction protocols that do not include a separate demineralization stage (e.g., Cleland & Vashishth, 2015; Hill et al, 2015) may be particularly well suited for the recovery of NCPs from fossils, and should be further explored.…”

“…For samples in which analysis of a separate demineralization fraction is not feasible, alternative methods that do not fractionate demineralization, or do not demineralize (e.g., Cleland & Vashishth, 2015; Hill et al, 2015), should be considered to avoid loss of identified protein diversity.…”

Peptide sequences from the first Castoroides ohioensis skull and the utility of old museum collections for palaeoproteomics

“…Two sets of fragments from the nasal turbinates (sample 1, 19.5 mg; sample 2, 19.8 mg) of NYSM VP-47 were homogenized with a Bead Ruptor 24 (Omni International) using the non-demineralization buffer from [20] to extract proteins. The turbinates were chosen because this skull has an apparent varnish on the entire exterior (figure 2) that does not appear to have been applied within the nasal cavity.…”