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Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways.


ABSTRACT: Quantitative measurement of the major regulatory proteins in signaling networks poses several technical challenges, including low abundance, the presence of post-translational modifications (PTMs), and the lack of suitable affinity detection reagents. Using the innate immune response (IIR) as a model signaling pathway, we illustrate the approach of stable isotope dilution (SID)-selected reaction monitoring (SRM)-mass spectrometry (MS) assays for quantification of low abundance signaling proteins. A work flow for SID-SRM-MS assay development is established for proteins with experimentally observed MS spectra and for those without. Using the interferon response factor (IRF)-3 transcription factor as an example, we illustrate the steps in high responding signature peptide identification, SID-SRM-MS assay optimization, and evaluation. SRM assays for normalization of IIR abundance to invariant housekeeping proteins are presented. We provide an example of SID-SRM assay development for post-translational modification (PTM) detection using an activating phospho-Ser modified NF-?B/RelA transcription factor, and describe challenges inherent in PTM-SID-SRM-MS assay development. Application of highly qualified quantitative, SID-SRM-MS assays will enable a systems-level approach to understanding the dynamics and kinetics of signaling in host cells, such as the IIR.

SUBMITTER: Zhao Y 

PROVIDER: S-EPMC3763905 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways.

Zhao Yingxin Y   Brasier Allan R AR  

Methods (San Diego, Calif.) 20130211 3


Quantitative measurement of the major regulatory proteins in signaling networks poses several technical challenges, including low abundance, the presence of post-translational modifications (PTMs), and the lack of suitable affinity detection reagents. Using the innate immune response (IIR) as a model signaling pathway, we illustrate the approach of stable isotope dilution (SID)-selected reaction monitoring (SRM)-mass spectrometry (MS) assays for quantification of low abundance signaling proteins  ...[more]

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