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Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling.

ABSTRACT: Peptide immunoaffinity enrichment coupled with targeted mass spectrometry is a quantitative approach for the robust and reproducible quantification of peptide analytes. The approach is capable of multiplexed quantification of peptides, including posttranslational modifications such as phosphorylation. Anti-peptide antibodies are used to enrich analytes and heavy stable isotope-labeled standards. The enriched peptides are directly measured by multiple reaction monitoring (MRM), a well-characterized quantitative mass spectrometry-based method. Quantification is performed by measuring the analyte (light) peptide response relative to the heavy standard, which is spiked at a known concentration. Here, we describe the methodology for multiplexed measurement of phosphorylated peptides on the ATM kinase and their nonmodified peptide analogs in cellular lysates. The method provides quantitative measurements of phospho-signaling and can be extended to a number of other phosphopeptides and sample types.

SUBMITTER: Whiteaker JR

PROVIDER: S-EPMC5555734 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project 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 × 10-9 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.

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Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling.

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