Chapter title |
A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses.
|
---|---|
Chapter number | 6 |
Book title |
Proteomics in Systems Biology
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Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3341-9_6 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3339-6, 978-1-4939-3341-9
|
Authors |
Annie Moradian, Tanya R. Porras-Yakushi, Michael J. Sweredoski, Sonja Hess |
Editors |
Jörg Reinders |
Abstract |
When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested. When guidelines that have been established in the pharmaceutical industry many decades ago are taken into account, setting up an MRM assay is relatively straightforward. Typically, proteotypic peptides with favorable mass spectrometric properties are synthesized with a heavy isotope for each protein that is to be monitored. Retention times and calibration curves are determined using triple-quadrupole mass spectrometers. The use of iRT peptide standards is both recommended and fully integrated into the bioinformatics pipeline. Digested biological samples are mixed with the heavy and iRT standards and quantified. Here we present a generic protocol for the development of an MRM assay. |
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