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

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....

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Veröffentlicht in:Molecular & cellular proteomics 2017-10, Vol.16 (10), p.1829-1849
Hauptverfasser: Hsiao, Yung-Chin, Chi, Lang-Ming, Chien, Kun-Yi, Chiang, Wei-Fan, Chen, Szu-Fan, Chuang, Yao-Ning, Lin, Shih-Yu, Wu, Chia-Chun, Chang, Ya-Ting, Chu, Lichieh Julie, Chen, Yi-Ting, Chia, Shu-Li, Chien, Chih-Yen, Chang, Kai-Ping, Chang, Yu-Sun, Yu, Jau-Song
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container_end_page 1849
container_issue 10
container_start_page 1829
container_title Molecular & cellular proteomics
container_volume 16
creator Hsiao, Yung-Chin
Chi, Lang-Ming
Chien, Kun-Yi
Chiang, Wei-Fan
Chen, Szu-Fan
Chuang, Yao-Ning
Lin, Shih-Yu
Wu, Chia-Chun
Chang, Ya-Ting
Chu, Lichieh Julie
Chen, Yi-Ting
Chia, Shu-Li
Chien, Chih-Yen
Chang, Kai-Ping
Chang, Yu-Sun
Yu, Jau-Song
description 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 × 10−9m 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 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.
doi_str_mv 10.1074/mcp.RA117.000147
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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 &lt; 2.82 × 10−9m could identify most clones with an immuno-capture recovery rate &gt;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 &lt;10, 10–50, and &gt;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. 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Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>2017 by The American Society for Biochemistry and Molecular Biology, Inc.</rights><rights>Copyright American Society for Biochemistry and Molecular Biology Oct 2017</rights><rights>2017 by The American Society for Biochemistry and Molecular Biology, Inc. 2017 The American Society for Biochemistry and Molecular Biology, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c475t-c5480db69a33e9b548a64a247b18c10d86ff3466f9e65aa825f98154211995213</citedby><cites>FETCH-LOGICAL-c475t-c5480db69a33e9b548a64a247b18c10d86ff3466f9e65aa825f98154211995213</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629267/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629267/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28821604$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hsiao, Yung-Chin</creatorcontrib><creatorcontrib>Chi, Lang-Ming</creatorcontrib><creatorcontrib>Chien, Kun-Yi</creatorcontrib><creatorcontrib>Chiang, Wei-Fan</creatorcontrib><creatorcontrib>Chen, Szu-Fan</creatorcontrib><creatorcontrib>Chuang, Yao-Ning</creatorcontrib><creatorcontrib>Lin, Shih-Yu</creatorcontrib><creatorcontrib>Wu, Chia-Chun</creatorcontrib><creatorcontrib>Chang, Ya-Ting</creatorcontrib><creatorcontrib>Chu, Lichieh Julie</creatorcontrib><creatorcontrib>Chen, Yi-Ting</creatorcontrib><creatorcontrib>Chia, Shu-Li</creatorcontrib><creatorcontrib>Chien, Chih-Yen</creatorcontrib><creatorcontrib>Chang, Kai-Ping</creatorcontrib><creatorcontrib>Chang, Yu-Sun</creatorcontrib><creatorcontrib>Yu, Jau-Song</creatorcontrib><title>Development of a Multiplexed Assay for Oral Cancer Candidate Biomarkers Using Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry</title><title>Molecular &amp; cellular proteomics</title><addtitle>Mol Cell Proteomics</addtitle><description>Oral cancer is one of the most common cancers worldwide, and there are currently no biomarkers approved for aiding its management. 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Chi, Lang-Ming ; Chien, Kun-Yi ; Chiang, Wei-Fan ; Chen, Szu-Fan ; Chuang, Yao-Ning ; Lin, Shih-Yu ; Wu, Chia-Chun ; Chang, Ya-Ting ; Chu, Lichieh Julie ; Chen, Yi-Ting ; Chia, Shu-Li ; Chien, Chih-Yen ; Chang, Kai-Ping ; Chang, Yu-Sun ; Yu, Jau-Song</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c475t-c5480db69a33e9b548a64a247b18c10d86ff3466f9e65aa825f98154211995213</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Animals</topic><topic>Antibodies, Monoclonal - chemistry</topic><topic>Antibodies, Monoclonal - immunology</topic><topic>Antibody Affinity - immunology</topic><topic>Antigens</topic><topic>Assaying</topic><topic>Biomarkers</topic><topic>Biomarkers, Tumor - analysis</topic><topic>Biomarkers, Tumor - metabolism</topic><topic>Body fluids</topic><topic>Cancer</topic><topic>Carcinoma, Squamous Cell - diagnosis</topic><topic>Coefficient of variation</topic><topic>Computer Simulation</topic><topic>Enrichment</topic><topic>Humans</topic><topic>Immunoassay</topic><topic>Limit of Detection</topic><topic>Mass spectrometry</topic><topic>Mass Spectrometry - methods</topic><topic>Mass spectroscopy</topic><topic>Mice</topic><topic>Monoclonal antibodies</topic><topic>Mouth Neoplasms - diagnosis</topic><topic>Multiplexing</topic><topic>Oral cancer</topic><topic>Patients</topic><topic>Peptides</topic><topic>Peptides - immunology</topic><topic>Proteomics - methods</topic><topic>Quantitation</topic><topic>Saliva</topic><topic>Saliva - chemistry</topic><topic>Scientific imaging</topic><topic>Spectroscopy</topic><topic>Target detection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hsiao, Yung-Chin</creatorcontrib><creatorcontrib>Chi, Lang-Ming</creatorcontrib><creatorcontrib>Chien, Kun-Yi</creatorcontrib><creatorcontrib>Chiang, Wei-Fan</creatorcontrib><creatorcontrib>Chen, Szu-Fan</creatorcontrib><creatorcontrib>Chuang, Yao-Ning</creatorcontrib><creatorcontrib>Lin, Shih-Yu</creatorcontrib><creatorcontrib>Wu, Chia-Chun</creatorcontrib><creatorcontrib>Chang, Ya-Ting</creatorcontrib><creatorcontrib>Chu, Lichieh Julie</creatorcontrib><creatorcontrib>Chen, Yi-Ting</creatorcontrib><creatorcontrib>Chia, Shu-Li</creatorcontrib><creatorcontrib>Chien, Chih-Yen</creatorcontrib><creatorcontrib>Chang, Kai-Ping</creatorcontrib><creatorcontrib>Chang, Yu-Sun</creatorcontrib><creatorcontrib>Yu, Jau-Song</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular &amp; 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subjects Animals
Antibodies, Monoclonal - chemistry
Antibodies, Monoclonal - immunology
Antibody Affinity - immunology
Antigens
Assaying
Biomarkers
Biomarkers, Tumor - analysis
Biomarkers, Tumor - metabolism
Body fluids
Cancer
Carcinoma, Squamous Cell - diagnosis
Coefficient of variation
Computer Simulation
Enrichment
Humans
Immunoassay
Limit of Detection
Mass spectrometry
Mass Spectrometry - methods
Mass spectroscopy
Mice
Monoclonal antibodies
Mouth Neoplasms - diagnosis
Multiplexing
Oral cancer
Patients
Peptides
Peptides - immunology
Proteomics - methods
Quantitation
Saliva
Saliva - chemistry
Scientific imaging
Spectroscopy
Target detection
title Development of a Multiplexed Assay for Oral Cancer Candidate Biomarkers Using Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry
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