title: Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.
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 (totaln= 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.
authors.affiliation: From the ‡Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; ytchen@mail.cgu.edu.tw.|§Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.|¶Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.|‖Department of Nephrology, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan.
authors.forename: Yi-Ting
authors.identifier: 0000-0002-0063-4965
authors.initials: YT
authors.lastname: Chen
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Hsiao-Wei
authors.initials: HW
authors.lastname: Chen
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Chun-Feng
authors.initials: CF
authors.lastname: Wu
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.|**Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Lichieh Julie
authors.initials: LJ
authors.lastname: Chu
authors.affiliation: ‡‡Department of Oral & Maxillofacial Surgery, Chi-Mei Medical Center, Liouying, Taiwan.|§§School of Dentistry, National Yang Ming University, Taipei, Taiwan.
authors.forename: Wei-Fang
authors.initials: WF
authors.lastname: Chiang
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.|¶¶Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.|‖‖Department of Otolaryngology - Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Chih-Ching
authors.initials: CC
authors.lastname: Wu
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.|¶Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.|**Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Jau-Song
authors.initials: JS
authors.lastname: Yu
authors.affiliation: ¶Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Cheng-Han
authors.initials: CH
authors.lastname: Tsai
authors.affiliation: **Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Kung-Hao
authors.initials: KH
authors.lastname: Liang
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.|¶Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.|‖‖Department of Otolaryngology - Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Yu-Sun
authors.initials: YS
authors.lastname: Chang
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Maureen
authors.initials: M
authors.lastname: Wu
authors.affiliation: §Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Ou Yang
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D002138:Calibration; D016022:Case-Control Studies; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D009062:Mouth Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D018307:Neoplasms, Squamous Cell / Q000175:diagnosis / Q000378:metabolism; D015203:Reproducibility of Results; D012471:Salivary Proteins and Peptides / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D012471:Salivary Proteins and Peptides