Chapter title |
Antibody-Based Capture of Target Peptides in Multiple Reaction Monitoring Experiments
|
---|---|
Chapter number | 7 |
Book title |
Mammary Stem Cells
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2519-3_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2518-6, 978-1-4939-2519-3
|
Authors |
Tommaso De Marchi, Eric Kuhn, Steven A. Carr, Arzu Umar |
Abstract |
Targeted quantitative mass spectrometry of immunoaffinity-enriched peptides, termed immuno-multiple reaction monitoring (iMRM), is a powerful method for determining the relative abundance of proteins in complex mixtures, like plasma or whole tissue. This technique combines 1,000-fold enrichment potential of antibodies for target peptides with the selectivity of multiple reaction monitoring mass spectrometry (MRM-MS). Using heavy isotope-labeled peptide counterparts as internal standards ensures high levels of precision. Further, LC-MRM-MS selectivity allows for multiplexing; antibodies recognizing different peptides can be added directly to a single mixture without subjecting to interferences common to other multiple antibody protein assays. Integrated extracted ion chromatograms (XIC) of product ions from endogenous unlabeled "light" peptide and stable isotope-labeled internal standard "heavy" peptides are used to generate a light/heavy peak area ratio. This ratio is proportional to the amount of peptide in the digestion mixture and can be used to estimate the concentration of protein in the sample. |
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