Empirical evaluation of bone extraction protocols.

The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and...

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Autores principales:	Timothy P Cleland, Kristyn Voegele, Mary H Schweitzer
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ee526e479cb14c7fb922065241dacc3c
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spelling	oai:doaj.org-article:ee526e479cb14c7fb922065241dacc3c2021-11-18T07:28:21ZEmpirical evaluation of bone extraction protocols.1932-620310.1371/journal.pone.0031443https://doaj.org/article/ee526e479cb14c7fb922065241dacc3c2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22348088/?tool=EBIhttps://doaj.org/toc/1932-6203The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on enzyme-linked immunosorbent assay and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extraction protocols tested on ostrich bone. For the immunological part of this study, we focused on collagen I, osteocalcin, and hemoglobin because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and hemoglobin is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining organics are chemically extracted. We found that the use of hydrochloric acid, rather than ethylenediaminetetraacetic acid, for demineralization resulted in the cleanest extractions because the acid was easily removed. In contrast, the use of ethylenediaminetetraacetic acid resulted in smearing upon electrophoretic separation, possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extracting bone proteins. One additional method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extraction analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extracting many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone.Timothy P ClelandKristyn VoegeleMary H SchweitzerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 2, p e31443 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Timothy P Cleland Kristyn Voegele Mary H Schweitzer Empirical evaluation of bone extraction protocols.
description	The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on enzyme-linked immunosorbent assay and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extraction protocols tested on ostrich bone. For the immunological part of this study, we focused on collagen I, osteocalcin, and hemoglobin because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and hemoglobin is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining organics are chemically extracted. We found that the use of hydrochloric acid, rather than ethylenediaminetetraacetic acid, for demineralization resulted in the cleanest extractions because the acid was easily removed. In contrast, the use of ethylenediaminetetraacetic acid resulted in smearing upon electrophoretic separation, possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extracting bone proteins. One additional method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extraction analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extracting many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone.
format	article
author	Timothy P Cleland Kristyn Voegele Mary H Schweitzer
author_facet	Timothy P Cleland Kristyn Voegele Mary H Schweitzer
author_sort	Timothy P Cleland
title	Empirical evaluation of bone extraction protocols.
title_short	Empirical evaluation of bone extraction protocols.
title_full	Empirical evaluation of bone extraction protocols.
title_fullStr	Empirical evaluation of bone extraction protocols.
title_full_unstemmed	Empirical evaluation of bone extraction protocols.
title_sort	empirical evaluation of bone extraction protocols.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/ee526e479cb14c7fb922065241dacc3c
work_keys_str_mv	AT timothypcleland empiricalevaluationofboneextractionprotocols AT kristynvoegele empiricalevaluationofboneextractionprotocols AT maryhschweitzer empiricalevaluationofboneextractionprotocols
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Empirical evaluation of bone extraction protocols.

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