Abstract

Aim: To identify differentially expressed proteins (DEPs) in dentinal fluid across a range of pulpal inflammatory stages—mild, moderate, severe pulpitis—using mass spectrometry. Methodology: This cross-sectional study analysed dentinal fluid from 60 patients categorised into healthy pulp (control), mild, moderate and severe pulpitis groups, based on Wolters' pulp diagnostic criteria. Pulp conditions were assessed through cold sensibility tests and radiographs, with inclusion limited to patients aged 12–40, who were systemically healthy and exhibited no advanced periodontal or apical pathology. Dentinal fluid was collected under aseptic conditions, stored at ?80°C and pooled into 12 representative samples for proteomic analysis. Proteins were extracted using lysis buffers and processed via LC–MS/MS with label-free quantification (LFQ) to identify DEPs. Enrichment analysis and protein–protein interaction (PPI) networks were conducted using Gene Ontology (GO), KEGG databases and STRING, with hub proteins identified using cytoscape. Statistical analysis employed Bayesian t-tests and linear models to evaluate protein expression, with a significance threshold of p < 0.05. Results: The severe pulpitis group exhibited the highest prevalence of systemic diseases (40%) compared with other groups (6.6%). LC–MS/MS identified 577 proteins, with 62 consistently quantified across the groups. The number of DEPs increased with inflammation severity, with 13 DEPs in severe pulpitis compared with controls. Principal component analysis (PCA) revealed partial separation between control and severe inflammation groups, with significant overlap between mild and moderate stages. Functional enrichment identified key biological pathways, including immune response, energy metabolism and structural integrity. Proteins such as cofilin-1, haemoglobin subunit alpha and peroxiredoxin-1 were upregulated in severe inflammation, while hornerin and myosin light chain 6 were downregulated. These findings highlight proteomic changes associated with pulpitis progression and identify potential prognostic biomarkers and therapeutic targets. Conclusions: This study identified distinct proteomic differences across pulpitis stages, with unique proteins in severe cases. These findings highlight novel biomarkers for advancing precise, cost-effective, point-of-care diagnostics and therapies, including multiplex platforms or ELISA assays. Proteomic analysis shows promise for understanding disease mechanisms and enabling personalised treatment strategies in endodontics. © 2025 British Endodontic Society. Published by John Wiley & Sons Ltd.