Identification of novel salivary candidate protein biomarkers for tuberculosis diagnosis

Abstract Background: The diagnosis of TB remains one of the major challenges in the control of the disease, due to limitations in the currently available diagnostic tests. There is an urgent need for new, accurate, rapid, and affordable diagnostic tests. The aim of the present study was to use mass spectrometry to identify new candidate TB diagnostic protein biomarkers in saliva obtained from individuals with TB, and patients with other respiratory diseases (ORD).Methods: Saliva samples were collected from 22 individuals who self-presented with symptoms requiring investigation for TB as part of a larger TB biomarker project. Eleven of the participants were finally diagnosed with TB using routine clinical, radiological and laboratory tests and 11 with ORD. Salivary proteins were concentrated and purified, followed by tryptic digestion. Peptides were analyzed using a QExactive Orbitrap MS coupled to a Dionex liquid chromatography system. Raw files were processed using MaxQuant software against the database of human proteins. Identified proteins were subjected to gene ontology and ingenuity pathway analysis for functional enrichment analysis.Results: We identified 1176 protein groups across all samples, of which 46 (3.91%) were contaminants, 12 (1.02%) were reverse hits and 170 (14.46%) were single-peptide protein groups. After removal of the contaminants, reverse hits and protein groups represented by single peptides, 26 of the remaining 948 proteins significantly discriminated individuals with TB from those with ORD after Benjamini Hochberg correction, with five of these proteins diagnosing TB with AUC ³ 0.80. A 5-protein biosignature comprising of P01011, Q8NCW5, P28072, A0A2Q2TTZ9 and Q99574 diagnosed TB with an AUC of 1.00 (95% CI, 1.00-1.00), sensitivity of 100% (95% CI, 76.2-100%) and specificity of 90.9% (95% CI, 58.7-99.8%) after leave-one-out cross validation. Conclusions: We identified novel salivary protein biomarkers and biosignatures with strong potential as TB diagnostic candidates. Our results are preliminary and require validation in larger studies.