Variability Assessment of 90 Salivary Proteins in Intraday and Interday Samples from Healthy Donors by Multiple Reaction Monitoring-Mass Spectrometry

Purpose: Saliva is an attractive sample source for the biomarker-based testing of several diseases, especially oral cancer. Here, we sought to apply multiplexed LC-MRM-MS to precisely quantify 90 disease-related proteins and assess their intra- and interindividual variability in saliva samples from healthy donors. Experimental design: We developed two multiplexed LC-MRM-MS assays for 122 surrogate peptides representing a set of disease-related proteins. Saliva samples were collected from 10 healthy volunteers at three different time points (Day 1 morning and afternoon, and Day 2 morning). Each sample was spiked with a constant amount of a ¹⁵ N-labeled protein and analyzed by MRM-MS in triplicate. Quantitative results from LC-MRM-MS were calculated by single-point quantification with reference to a known amount of internal standard (heavy peptide). Results: The CVs for assay reproducibility and technical variation were 13 and 11%, respectively. The average concentrations of the 99 successfully quantified proteins ranged from 0.28 ± 0.58 ng mL ⁻¹ for profilin-2 (PFN2) to 8.55 ±8.96 μg mL ⁻¹ for calprotectin (S100A8). For the 90 proteins detectable in >50% of samples, the average CVs for intraday, interday, intraindividual, and interindividual samples were 38%, 43%, 45%, and 69%, respectively. The fluctuations of most target proteins in individual subjects were found to be within ± twofold. Conclusions and clinical relevance: Our study elucidated the intra- and interindividual variability of 90 disease-related proteins in saliva samples from healthy donors. The findings may facilitate the further development of salivary biomarkers for oral and systemic diseases.