Solid Digestion of Demineralized Bone as a Method To Access Potentially Insoluble Proteins and Post-Translational Modifications

Bone proteomics is an expanding field for understanding protein changes associated with disease as well as characterizing and detecting proteins preserved in fossil bone. Most previous studies have utilized a protocol with demineralization and extraction approach to isolate and characterize proteins from bone. Through near-complete EDTA demineralization, followed by solid digestion of the remaining bone pseudomorph, a total of 92 protein accessions were detected from dog bone. In the EDTA, 14 unique proteins were found, including osteocalcin, an important bone protein. Osteocalcin was not found in the solid digestion samples, demonstrating the importance of examining the demineralization supernatant. The solid-digestion samples were analyzed both with (11 unique accessions) and without (16 unique accessions) alkylation, resulting in a total of 78 protein accessions. In addition to the diversity of proteins detected, various post-translational modifications were observed, including phosphorylation and glycosylation. The solid-digestion approach will allow for characterization of proteins that are insoluble and would otherwise be missed by traditional bone protein extraction alone. All data are available at ftp://massive.ucsd.edu/MSV000081399

SDS-PAGE gel of moa extractions.

<p>Arrowheads indicate faint bands visible through the smearing.</p

Summary of Franzén and Heinegård methods.

<p>*Dialysis and lyophilization is abbreviated D/L. Precip represents chloroform∶methanol∶water precipitation performed on half of the supernatant. Volumes correspond to the number of milliliters of buffer multiplied by the grams of bone powder.</p

Moa Ammonium Bicarbonate-1

Moa ammonium bicarbonate extraction run #1 on Orbitrap X

Summary of Rabilloud method, Craig and Collins method, Embery method, and Schweitzer method.

<p>*Dialysis and lyophilization is abbreviated D/L. Precip represents chloroform∶methanol∶water precipitation performed on half of the supernatant. Volumes correspond to the number of milliliters of buffer multiplied by the grams of bone powder.</p

Yields from ∼1.3 g of bone powder of each extraction protocol described in Table 1.

<p>B.D. refers to below the limit of detection for the balance used (0.1 mg). Total values correspond to lyophilized samples only and are calculated by addition of each step of an individual protocol.</p

Moa enzyme-linked immunosorbent assay results showing means plus or minus one standard deviation.

<p>Values correspond to absorbance at 405 nm. − represents no detected absorption. + represents at least two times the average absorbance of buffer control.</p

Summary of Jiang method and Buckley method.

<p>*Dialysis and lyophilization is abbreviated D/L. Volumes correspond to the number of milliliters of buffer multiplied by the grams of bone powder.</p

Moa HCl control

Moa HCl extraction buffer control on Orbitrap X

Moa Ammonium Bicarbonate-2

Moa ammonium bicarbonate extraction run #2 on Orbitrap X