Development of a Multiplexed Assay for Oral Cancer Candidate Biomarkers Using Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry

Yung-Chin Hsiao,Lang-Ming Chi,Kun-Yi Chien,Wei-Fan Chiang,Szu-Fan Chen,Yao-Ning Chuang,Shih-Yu Lin,Chia-Chun Wu,Ya-Ting Chang,Lichieh Julie Chu,Yi-Ting Chen,Shu-Li Chia,Chih-Yen Chien,Kai-Ping Chang,Yu-Sun Chang,Jau-Song Yu

doi:10.1074/mcp.ra117.000147

Abstract

Oral cancer is one of the most common cancers worldwide, and there are currently no biomarkers approved for aiding its management. Although many potential oral cancer biomarkers have been discovered, very few have been verified in body fluid specimens in parallel to evaluate their clinical utility. The lack of appropriate multiplexed assays for chosen targets represents one of the bottlenecks to achieving this goal. In the present study, we develop a peptide immunoaffinity enrichment-coupled multiple reaction monitoring-mass spectrometry (SISCAPA-MRM) assay for verifying multiple reported oral cancer biomarkers in saliva. We successfully produced 363 clones of mouse anti-peptide monoclonal antibodies (mAbs) against 36 of 49 selected targets, and characterized useful mAbs against 24 targets in terms of their binding affinity for peptide antigens and immuno-capture ability. Comparative analyses revealed that an equilibrium dissociation constant (KD ) cut-off value < 2.82 × 10-9 m could identify most clones with an immuno-capture recovery rate >5%. Using these mAbs, we assembled a 24-plex SISCAPA-MRM assay and optimized assay conditions in a 25-μg saliva matrix background. This multiplexed assay showed reasonable precision (median coefficient of variation, 7.16 to 32.09%), with lower limits of quantitation (LLOQ) of <10, 10-50, and >50 ng/ml for 14, 7 and 3 targets, respectively. When applied to a model saliva sample pooled from oral cancer patients, this assay could detect 19 targets at higher salivary levels than their LLOQs. Finally, we demonstrated the utility of this assay for quantification of multiple targets in individual saliva samples (20 healthy donors and 21 oral cancer patients), showing that levels of six targets were significantly altered in cancer compared with the control group. We propose that this assay could be used in future studies to compare the clinical utility of multiple oral cancer biomarker candidates in a large cohort of saliva samples.

Highlights

We report the production and characterization of anti-peptide mouse monoclonal antibodies and the subsequent successful development of a 24-plex SISCAPA-MRM assay for quantifying multiple oral squamous cell carcinomas (OSCCs) candidate biomarkers in saliva samples
Signature peptides of prioritized targets were selected for mouse monoclonal antibodies (mAbs) production and synthesis of stable isotope-labeled (SIS) peptides, and the sequence of the synthetic peptides were confirmed by MALDI-TOF/TOF MS analysis
Applying a receiver operating characteristic (ROC) analysis to evaluate the power of ka, kd and KD to predict mAbs with higher peptide-capture ability, we found that the area under the curve (AUC) values of KD, kd and ka were 0.91, 0.86, and 0.68, respectively (Fig. 4B), indicating that KD and, to a lesser extent, kd, were good predictors for selecting mAbs suitable for SISCAPA assay development

Summary

Introduction

Comparative analyses revealed that an equilibrium dissociation constant (KD) cut-off value < 2.82 ؋ 10؊9 M could identify most clones with an immuno-capture recovery rate >5% Using these mAbs, we assembled a 24-plex SISCAPA-MRM assay and optimized assay conditions in a 25-␮g saliva matrix background. When applied to a model saliva sample pooled from oral cancer patients, this assay could detect 19 targets at higher salivary levels than their LLOQs. we demonstrated the utility of this assay for quantification of multiple targets in individual saliva samples (20 healthy donors and 21 oral cancer patients), showing that levels of six targets were significantly altered in cancer compared with the control group. The World Health Organization predicts that the incidence of oral cavity cancer will continue to increase worldwide for the several decades, especially in Asia, because of distinct cultural practices such as betel-quid chewing [6, 7]. No biomarkers that meet this need are approved by official health agencies in endemic areas [15]

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Conclusion