Shotgun Proteomics of Human Dentin with Different Prefractionation Methods

Abstract

Human dentin is not only a composite material of a collagenous matrix and mineral to provide strength and elasticity to teeth, but also a precious reservoir full of bioactive proteins. They are released after demineralization caused by bacterial acids in carious lesions, by decalcifying irrigants or dental materials and they modulate tissue responses in the underlying dental pulp. This work describes a first-time analysis of the proteome of human dentin using a shotgun proteomic approach that combines three different protein fractionation methods. Dentin matrix proteins were extracted by EDTA and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), OFFGEL isoelectric focusing (IEF) or strong cation exchange chromatography (SCX). Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 813 human proteins with high confidence, however, isoelectric focusing turned out to be the most beneficial prefractionation method. All Proteins were categorized based on the PANTHER system and representation analysis revealed 31 classes and subclasses to be overrepresented. The acquired knowledge provides a comprehensive insight into the number of proteins in human dentin as well as their physiological and pathological functions. Thus, the data presented paves the way to the analysis of specific functions of dentin matrix proteins in vivo and their potential in tissue engineering approaches to regenerate dental pulp.

Figure 1

The Venn diagram shows overlapping proteins detected in human dentin by mass spectrometry and combination of different prefractionation methods: isoelectric focusing (IEF), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS) and strong cation exchange chromatography (SCX).

Figure 2

Analysis of human dentin proteome by mass spectrometry and combination of three different prefractionation methods. (a) Number of repeatedly detected proteins (390) with the best Mascot score in each prefractionation group. (b) Medians and 25–75% percentiles of Mascot scores achieved after IEF, SDS-PAGE and SCX. Each group contains proteins that were uniquely detected or provided the best Mascot score at repeated detection. Asterisks indicate significant differences between groups (P ≤ 0.05). (c) Pie charts show the number of single peptides detected in mass spectrometry by different prefractionation methods.

Figure 3

Protein classes detected in human dentin at the most general level and, in parentheses, the number of proteins in each family.