Proteomic analysis of human dentin matrix extracts by peptide-level high-pH reverse-phase fractionation

Proteomics research has increasingly focused on human cells, tissues, and fluids; however, comprehensive data regarding dental tissues remain limited. Dentin, a mineralized component of the tooth, contains structural proteins and bioactive molecules that, upon release, can modulate pulp cell activity and facilitate regeneration. Therefore, an understanding of the protein composition of dentin is of considerable importance. Previous studies have reported a limited number of proteins, and technical difficulties have often hindered replication. Furthermore, traditional extraction methods rely on strong acids, which are not clinically relevant. In this technical note, we introduce a workflow that combines EDTA-based dentin extraction under clinically relevant conditions with peptide-level fractionation using high-pH reverse-phase chromatography. This approach was compared against unfractionated samples (control), protein-level fractionation via SDS-PAGE, and peptide-level fractionation utilizing strong cation exchange (SCX), all followed by LC-MS/MS analysis. This workflow facilitated the identification of 514 proteins, compared with 238 (unfractionated control), 428 (SDS-PAGE), and 193 (SCX). High-pH reverse-phase chromatography contributed 217 unique identifications, surpassing the number of unique fractions identified through the other techniques. Although this methodology has been utilized in other protein systems, it has not previously been applied to dentin matrix extracts and represents a promising approach for enhancing the comprehensiveness of protein discovery.