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.