In this seminar, we outlined the robust and sensitive quantification of peptides using automated immunoaffinity enrichment, stable isotope labeled peptides and nanoLC-MRM-MS.
Targeted mass spectrometry is routinely employed to identify and quantify peptides and proteins in complex matrices such as tissues and plasma. Immunoaffinity enrichment of peptides coupled with analysis by stable isotope dilution multiple reaction mass spectrometry has been shown to have analytical performance and detection limits suitable for many biomarker verification studies and biological applications. Prior studies have shown that anti-peptide antibodies can be multiplexed up to 50 in a single assay without significant loss of performance.
However, achieving even higher multiplex levels is relevant to all studies involving precious biological material as this minimizes the amount of sample that must be consumed to measure a given set of analytes and reduces the assay cost per analyte. In this talk, we described the development and performance characterization of an automated immunoaffinity workflow up to multiplex levels of 172. We also detailed the application of a 120-plex iMRM assay to probe for protein concentration trends in plasma from patients undergoing a therapeutic planned myocardial infarction to identify biomarkers of cardiovascular disease.
Eric Kuhn is a Research Scientist at the Broad Institute in the Proteomics and Biomarker Discovery platform. He is an expert in peptide quantitation assays that use stable isotope labeled standards and LC-MRM-MS. He has designed and implemented enrichment strategies, such as immunodepletion, multidimensional chromatography and peptide immunoprecipitation, to increase sensitivity and precision. The application of these assays has been an integral part of biomarker discovery programs in ovarian cancer, breast cancer, rheumatoid arthritis and cardiovascular disease for more than 15 years. He has co-authored guidance documents that describe peptide standard specifications and targeted peptide measurement best practices. Eric has a BS in Biochemistry from University of New Hampshire. His has developed analytical methods at Dupont-NEN, AutoImmune, and Millennium Pharmaceuticals.
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