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

Abstract Saliva offers a non⍰invasive, low⍰cost, and patient⍰friendly matrix for biomarker discovery. Affinity⍰based proteomic technologies such as the Proximity Extension Assay (PEA) are increasingly being adopted for large⍰scale biomarker studies, yet they remain underexplored in saliva. This study applied the Olink Explore High⍰Throughput (HT) PEA platform to profile approximately 5,400 proteins in saliva samples collected from donors representing periodontal health, gingivitis, advanced periodontitis (baseline and 3⍰months post⍰treatment), and edentulism. Saliva from 68 donors was analysed, and all samples passed Olink’s quality⍰control procedures, with only 17 of 5,416 assays failing. Forty⍰one 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 post⍰treatment 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 platform’s minimal sample⍰volume requirements and scalability present strong potential for future saliva⍰based biomarker discovery and translational research.