Abstract
Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research.
Highlights
Multiple reaction monitoring (MRM)1, known as selected reaction monitoring (SRM), of peptides is a growing
We report an optimized workflow for precise quantitation of 56 saliva proteins in a single run using LCMRM/MS coupled with the use of stable isotope-labeled standard (SIS) peptides
Clinical saliva samples were collected for precise quantification of 56 target proteins by LC-MRM/MS for evaluation as Oral squamous cell carcinoma (OSCC) biomarkers
Summary
Multiple reaction monitoring (MRM), known as selected reaction monitoring (SRM), of peptides is a growing. Quantification of Salivary Proteins as Oral Cancer Biomarker technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplexcapable than antibody-based techniques, such as enzymelinked immunosorbent assays (ELISAs) [1,2,3,4]. In this MRMbased technique, which incorporates liquid chromatography with mass spectrometry detection (LC-MRM/MS), specific transitions of precursors selected in Q1 to fragment ions selected in Q3 are monitored using a triple-quadrupole MS instrument, generating signals for quantification. To demonstrate the potential clinical application of this workflow, we evaluated its analytical performance and quantitation ability in two sample sets and compared them between demographically matched control and oral cancer groups for verifying the clinical utility
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