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Antibody-based capture of target peptides in multiple reaction monitoring experiments

De Marchi, Tommaso LU ; Kuhn, Eric; Carr, Steven A. and Umar, Arzu (2015) In Methods in Molecular Biology 1293. p.35-123
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... (More)

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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author
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
keywords
Mass Spectrometry, Peptides, Proteomics, Journal Article, Research Support, Non-U.S. Gov't
in
Methods in Molecular Biology
volume
1293
pages
35 - 123
publisher
Humana Press
external identifiers
  • scopus:84930655255
DOI
10.1007/978-1-4939-2519-3_7
language
English
LU publication?
no
id
e2e5e3de-82ea-44d5-b192-3b796c1a70ff
date added to LUP
2017-06-27 14:28:22
date last changed
2017-07-23 05:30:10
@inbook{e2e5e3de-82ea-44d5-b192-3b796c1a70ff,
  abstract     = {<p>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.</p>},
  author       = {De Marchi, Tommaso and Kuhn, Eric and Carr, Steven A. and Umar, Arzu},
  keyword      = {Mass Spectrometry,Peptides,Proteomics,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  pages        = {35--123},
  publisher    = {Humana Press},
  series       = {Methods in Molecular Biology},
  title        = {Antibody-based capture of target peptides in multiple reaction monitoring experiments},
  url          = {http://dx.doi.org/10.1007/978-1-4939-2519-3_7},
  volume       = {1293},
  year         = {2015},
}