Affinity Proteomics for Saliva Biomarker Discovery Using High-Throughput Proximity Extension Assay

Saliva offers a noninvasive, lowcost, and patientfriendly matrix for biomarker discovery. Affinitybased proteomic technologies such as the Proximity Extension Assay (PEA) are increasingly being adopted for largescale biomarker studies, yet they remain underexplored in saliva. This study applied the Olink Explore HighThroughput (HT) PEA platform to profile approximately 5,400 proteins in saliva samples collected from donors representing periodontal health, gingivitis, advanced periodontitis (baseline and 3months posttreatment), and edentulism. Saliva from 68 donors was analysed, and all samples passed Olinks qualitycontrol procedures, with only 17 of 5,416 assays failing. Fortyone percent of proteins were detected above the limit of detection, demonstrating substantial assay sensitivity in this biofluid. Principal component analysis revealed clear compositional differences between clinical groups, with posttreatment periodontitis samples clustering more closely with health than baseline disease. Pairwise group comparisons identified hundreds of differentially abundant proteins, with consistently more proteins increased than decreased relative to health. This study demonstrates, for the first time, that Olink HT can robustly measure thousands of proteins in saliva with high data quality and biologically meaningful discrimination between periodontal states. The platforms minimal samplevolume requirements and scalability present strong potential for future salivabased biomarker discovery and translational research.