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.