Proteomic Analysis of Human Dentine Matrix Extracts by Peptide-Level High-pH Reversed-Phase Fractionation

Proteomics research has increasingly focused on human cells, tissues, and fluids; however, comprehensive data on dental tissues remain limited. Dentine, a mineralized component of teeth, contains structural proteins and bioactive molecules that can modulate pulp cell activity and support regeneration when released. Understanding its protein composition is therefore essential. Previous studies have identified relatively few dentine proteins, and technical challenges have hindered reproducibility. Traditional extraction methods also rely on strong acids that lack clinical relevance. In this technical note, we introduce a workflow combining EDTA-based dentine extraction under clinically relevant conditions with peptide-level fractionation using high-pH reversed-phase chromatography. This approach was compared with unfractionated samples, SDS-PAGE protein-level fractionation, and strong cation exchange (SCX) peptide-level fractionation, all followed by LC-MS/MS. Data are available via ProteomeXchange (PXD070849). This workflow enabled the identification of 514 proteins compared with 238 (unfractionated), 428 (SDS-PAGE), and 193 (SCX). High-pH reversed-phase chromatography contributed 217 unique identifications, exceeding those from other techniques. Although used in other proteomic systems, this methodology has not previously been applied to dentine matrix extracts and represents a promising approach for improving protein discovery.