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A method for whole protein isolation from human cranial bone.

Authors
  • Lyon, Sarah M1
  • Mayampurath, Anoop2
  • Rogers, M Rose3
  • Wolfgeher, Donald J4
  • Fisher, Sean M1
  • Volchenboum, Samuel L2
  • He, Tong-Chuan3
  • Reid, Russell R5
Type
Published Article
Journal
Analytical Biochemistry
Publisher
Elsevier
Publication Date
Dec 15, 2016
Volume
515
Pages
33–39
Identifiers
DOI: 10.1016/j.ab.2016.09.021
PMID: 27677936
Source
Medline
Keywords
License
Unknown

Abstract

The presence of the dense hydroxyapatite matrix within human bone limits the applicability of conventional protocols for protein extraction. This has hindered the complete and accurate characterization of the human bone proteome thus far, leaving many bone-related disorders poorly understood. We sought to refine an existing method of protein extraction from mouse bone to extract whole proteins of varying molecular weights from human cranial bone. Whole protein was extracted from human cranial suture by mechanically processing samples using a method that limits protein degradation by minimizing heat introduction to proteins. The presence of whole protein was confirmed by western blotting. Mass spectrometry was used to sequence peptides and identify isolated proteins. The data have been deposited to the ProteomeXchange with identifier PXD003215. Extracted proteins were characterized as both intra- and extracellular and had molecular weights ranging from 9.4 to 629 kDa. High correlation scores among suture protein spectral counts support the reproducibility of the method. Ontology analytics revealed proteins of myriad functions including mediators of metabolic processes and cell organelles. These results demonstrate a reproducible method for isolation of whole protein from human cranial bone, representing a large range of molecular weights, origins and functions.

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