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.

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

“…We were able to identify 524 proteins with 1397 peptides (Supporting Information ). Similar to other reports , we have got 76% of peptides (1061 peptides) in unique fractions (Figure A). Figures C shows the unique peptides (peptides detected in only one fraction) distribution per fractions.…”

“…In its most successful application, IEF is used as a first dimension of two dimensional electrophoretic systems, as reported since 1975 [5][6][7][8]. For shotgun proteomics, protein separation by IEF has been used as a pre-fractionation step, previous to the LC-MS/MS analysis [9][10][11][12]. For this aim, several instrument designs based on IEF fundamentals have been developed.…”

Cell-Free Approaches for Dental Pulp Tissue Engineering

“…However, methodical limitations of the applied techniques, especially regarding protein isolation and preparation, restricted protein detectability. A recent mass spectrometry analysis of dentin matrix proteins after prefractionation by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cation exchange chromatography detected a total amount of 813 proteins 31 . These were assigned to various protein classes according to the PANTHER (protein annotation through evolutionary relationship) system.…”

Large‐scale production, purification, and function of a tumor multi‐epitope vaccine: Peptibody with bFGF/VEGFA

“…Moreover, metal ions are banned from human vaccine; thus, columns chelated metal ions are out of our consideration [21]. In the industrial production, cation-exchange chromatography, an inexpensive column with high capacity, is widely used to catch recombinant protein with positive charge from soluble fractions and other endogenous proteins with negative charge will flow through in this column [22]. Hydrophobic chromatography is employed for further purification when the hydrophobic groups are exposed in high salinity [23].…”