Antibody-based capture of target peptides in multiple reaction monitoring experiments.
Authors | |
Keywords | |
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. |
Year of Publication | 2015
|
Journal | Methods Mol Biol
|
Volume | 1293
|
Pages | 123-35
|
Date Published | 2015
|
ISSN | 1940-6029
|
URL | |
DOI | 10.1007/978-1-4939-2519-3_7
|
PubMed ID | 26040685
|
Links |