A Novel Approach to Protein Analysis in Hard Tissues

Abstract

This preliminary study is aimed at a new approach in the protein analysis of hard tissues. Analytical methods that have been used for the identification of proteins in bones and teeth always require one critical step -demineralization. This chemical treatment is responsible for loss of proteins and it also negatively influences the possibility of protein quantification. The method of peptide mass mapping described in this paper facilitates a gentle releasing of peptides from the hard tissues without the loss of qualitative and quantitative information. Using this method led to identification of proteins from chicken thighbone, human jawbone and teeth, which proves the feasibility of this method.

Mass Spectrometry-Based Proteomics of Poly(methylmethacrylate)-Embedded Bone

“…Between 16 and 57 proteins were identified in the ‘T_cal’ samples, and between 109 and 208 proteins were identified in the ‘T_decal’ samples that were decalcified prior to protein extraction. These numbers are notably lower than those obtained from soft tissues, yet align with currently reported protein IDs from MS analyses of bone using nonfractionated and label-free DDA MS settings. ,− ,,, Although decalcification slightly increased protein coverage, both methods have less than 35% overlap between all replicates and thus demonstrate poor reproducibility (Figure B).…”

“…These numbers are notably lower than those obtained from soft tissues, yet align with currently reported protein IDs from MS analyses of bone using nonfractionated and label-free DDA MS settings. 32,[34][35][36]38,58,59 Although decalcification slightly increased protein coverage, both methods have less than 35% overlap between all replicates and thus demonstrate poor reproducibility (Figure 2B).…”

“…Although many combinations of the sample preparation steps have been reported, the numbers of identified proteins from bone samples are mostly confined to the double-digit range. 32,[34][35][36]38 Higher coverage has only been achieved when protein extraction processes were extended over several days or when extraction steps were performed sequentially and multiple fractions were collected. 31,37,39,40 Fractionation strategies are commonly used for samples with wide dynamic ranges in protein abundances as they reduce sample complexity and thereby favor the detection of low abundant proteins.…”

“…Numerous protocols have been proposed that address the technical challenges of protein extraction from bone. These protocols typically involve homogenization either by grinding bone samples to fine powders or using bead-based homogenizers in the presence of extraction buffers. − Decalcification is often applied to dissolve the inorganic bone mineral prior to protein solubilization using a variety of reagents, such as hydrochloric acid (HCl), sodium ethylenediaminetetraacetic acid (EDTA), or ammonium EDTA. Subsequent protein digestion is primarily performed in solution.…”

“…Subsequent protein digestion is primarily performed in solution. Although many combinations of the sample preparation steps have been reported, the numbers of identified proteins from bone samples are mostly confined to the double-digit range. ,− , Higher coverage has only been achieved when protein extraction processes were extended over several days or when extraction steps were performed sequentially and multiple fractions were collected. ,,, …”