3 years ago

Variability Assessment of 90 Salivary Proteins in Intra-Day and Inter-Day Samples from Healthy Donors by Multiple Reaction Monitoring-Mass Spectrometry

Yao-Ning Chuang, Lang-Ming Chi, Szu-Fan Chen, Yu-Sun Chang, Yi-Ting Chen, Kun-Yi Chien, Lichieh Julie Chu, Shih-Yu Lin, Jau-Song Yu, Wei-Fan Chiang, Yung-Chin Hsiao, Kai-Ping Chang, Chih-Yen Chien, Wei-Shun Wang, Ya-Ting Chang
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 inter-individual 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 15N-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 for profilin-2 (PFN2) to 8.55 ± 8.96 μg/mL for calprotectin (S100A8). For the 90 proteins detectable in > 50% of samples, the average CVs for intra-day, inter-day, intra-individual, and inter-individual samples were 38%, 43%, 45%, and 69%, respectively. The fluctuations of most target proteins in individual subjects were found to be within ± two-fold. Conclusions and clinical relevance Our study elucidated the intra- and inter-individual 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. This article is protected by copyright. All rights reserved

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/prca.201700039

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