Empirical Evaluation of Bone Extraction Protocols

Abstract

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.

Bone protein extraction without demineralization using principles from hydroxyapatite chromatography

“…Because extraction remains at a low level of total yield for extant bone [24; 27], bone protein extraction remains a major challenge in fully understanding the proteome of bone beyond the isolation and characterization of individual proteins [34; 35; 36; 37] Using methodology derived from hydroxyapatite chromatography for elution, we have developed a small scale technique to extract protein from extant bone that has yields higher than other small scale, previously reported non-demineralization and demineralization extractions (Fig. 3).…”

“…However, because bone is mineralized, analyzing the protein content in bone is more challenging than other non-mineralized tissue. In particular, protein extraction protocols rely on demineralization of bone followed by protein solubilization (reviewed in [24]). Consequently, the protocols are typically slow, taking days to weeks to perform [24], or may result in unknown breakdown of proteins by hydrolysis.…”

“…In particular, protein extraction protocols rely on demineralization of bone followed by protein solubilization (reviewed in [24]). Consequently, the protocols are typically slow, taking days to weeks to perform [24], or may result in unknown breakdown of proteins by hydrolysis. Through these traditional extraction protocols, ~1% or less of the original bone mass is extracted [24], most of which is composed of collagen I.…”

Biologically and diagenetically derived peptide modifications in moa collagens

“…Cortical bone fragments from an indeterminate moa specimen (MOR OST-255; 800-1000 years old [24]) were extracted as described by Cleland et al [23]. Briefly, approximately 1 g of cortical bone was demineralized in 9 ml 0.6 M HCl for 4 h at room temperature; the pellet was washed with sterile, deionized water and fragments were further extracted using 50 mM ammonium bicarbonate at 658C for 5 h. The HCl fraction was dialyzed against water (2000 MWCO Slide-A-Lyzer Cassettes; Pierce) for 4 days at 48C, then lyophilized to completion.…”

“…Because of the availability of exceptionally preserved specimens, DNA recovered from various moa specimens has been used to differentiate species [20,21] and estimate rates of DNA degradation [22]. However, few complementary studies of moa proteins have been attempted [3,23].…”