| Metadata |
| datasetIdentifier | PASS00910 |
| datasetType | SRM |
| submitter | Yung-Chin Hsiao <hschin@mail.cgu.edu.tw> |
| submitter_organization | Molecular Medicine Research Center, Chang Gung University |
| lab_head_full_name | Jau-Song Yu |
| lab_head_email | yusong@mail.cgu.edu.tw |
| lab_head_organization | Department of Cell and Molecular Biology, College of Medicine, Chang Gung University |
| lab_head_country | Taiwan |
| datasetTag | 24plex_iMRM |
| datasetTitle | SISCAPA-MRM assay for oral cancer biomarker verification |
| publicReleaseDate | 2017-09-19 00:00:00 |
| finalizedDate | 2017-09-19 20:05:52 |
| summary | 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 simultaneously in body fluid specimens 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-g 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. |
| contributors | Yung-Chin Hsiao, Lang-Ming Chi, Kun-Yi Chien, Wei-Fang Chiang, Szu-Fan Chen, Yao-Ning Chuang, Shih-Yu Lin, Chia-Chun Wu, Lichieh Julie Chu, Yi-Ting Chen, Shu-Ti Chiou, Shu-Li Cha, Kai-Ping Chang, Yu-Sun Chang, and Jau-Song Yu |
| publication | Hsiao, YC, Chi, LM, Chien, KY, Chiang, WF, Chen, SF, Chuang, YN, Lin, SY, Wu, CC, Chu, LJ, Chen, YT, Chiou, ST, Cha, SL, Chang, KP, Chang, YS, and Yu, JS, Development of a multiplexed assay for oral cancer candidate biomarkers using peptide immunoaffinity enrichment and targeted mass spectrometry, Mol Cell Proteomics, submitted |
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| treatment | |
| extraction | Saliva samples from healthy controls and OSCC patients were collected in two hospitals (Chi-Mei Medical Center, Liouying, Taiwan and Chang Gung Memorial Hospital, Linkou, Taiwan) and processed as described previously (52). The donors avoided eating, drinking, smoking, and using oral hygiene products for at least 1 hour before collection. The obtained saliva samples were centrifuged at 3,000 g for 15 minutes at 4°C. The resulting supernatant was collected, treated with a protease inhibitor cocktail (Sigma, MO, US), and stored in aliquots at -80°C. |
| separation | A KingFisher magnetic particle processor was used for automated handling of the transfer of magnetic beads between plates (plates 1 to 7). The wells of plate 1 contained 120 μl of protein G magnetic beads (GE Healthcare) and 80 μl of PBS plus 0.03% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; USB Corp., OH, US). The beads in plate 1 were washed for 5 minutes, transferred to plate 2 containing 24 mouse monoclonal antibodies against 24 analytes (1 μg antibody for each analyte), and incubated for 1 hour. Mouse IgGs bound to protein G magnetic beads were captured by the magnetic probe of the processor, transferred to plate 3 containing 25 μg of trypsin-digested saliva samples and 100 fmol of spiked SIS peptides, and then incubated in plate 3 for 2 hours. IgG-captured beads were then washed three times by sequentially transferring to plates 4, 5 and 6, which contained 200 μl of PBS (plates 4 and 5) or 0.1x PBS (plate 6). The total wash time through plates 4 to 6 was about 5 minutes. The captured peptides were then eluted in plate 7 containing 50 μl of 5% acetic acid (J.T. Baker, NJ, US) and 70% ACN. |
| digestion | Saliva samples containing equal amounts of protein were subjected to a tryptic digestion protocol as previously described (53). Briefly, 25 μg of protein diluted with an appropriate amount of 25 mM ammonium bicarbonate (Sigma, MO, US) was mixed with 40 μl of 20% (w/v) sodium deoxycholate (DOC; Sigma) and then boiled for 5 minutes. The denatured proteins were reduced by incubating with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Sigma) at 60°C for 30 minutes and then alkylated by incubating with 10 mM iodoacetamide (Sigma) at 37°C for 30 minutes in the dark. The sample was then diluted 3-fold with 0.1 M ammonium bicarbonate and digested with 4 μg trypsin (Agilent, CA, US) at 37°C overnight. Digestion was stopped by boiling for 15 minutes, followed by addition of 4 μg trypsin inhibitor (Sigma). Afterward, DOC was precipitated by adding 0.1% trifluoroacetic acid (TFA; Alfa Aesar, MA, US) and 0.4% formic acid (FA) to the digests, and the precipitated DOC was removed by centrifugation at 15,000 g for 10 minutes at room temperature. The supernatants were then desalted with solid-phase extraction (SPE) equipment using Waters Oasis HLB 96-well Elution plates (2 mg) (Waters, MA, US). |
| acquisition | MRM acquisition methods were constructed using three selected transitions (Q1/Q3 pairs) per peptide, as shown in Table S2, and each transition was monitored within a 6-minute scheduled MRM detection window. Optimization of data acquisition parameters (collision energy, CE; declustering potential, DP; entrance potential, EP; collision entrance potential, CEP; and collision exit potential, CXP) were essentially performed as previously described (50), with the following modifications: ion spray voltage, 2300 V; curtain gas setting, 20 psi (UHP nitrogen); interface heater temperature, 150°C; and MS operating pressure, 3.5 × 10-5 Torr; Q1 and Q3 were set to unit resolution (0.6–0.8 Da full width at half height) |
| informatics | All MRM data were processed using the Skyline (MacCoss Lab Software [39]. The mass list of transitions for measuring target peptide sequences was preloaded, and the peak areas of the selected target peptides were extracted for calculation. All of the retention times and transition profiles for the endogenous and exogenously spiked heavy peptides were manually checked to ensure that they were internally consistent. The endogenous peptide-specific peaks were identified by comparison with the exogenously added 13C/15N-labeled peptides. The concentration of each 13C/15N-labeled peptide was known, allowing the target protein concentration in each sample to be determined from the observed peak area ratios. |
| instruments | AB SCIEX QTRAP 5500 |
| species | Human |
| massModifications | K+8.014199, R+10.008269 |
