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

Yingxin Zhao, Allan R. Brasier

Research output: Contribution to journalArticle

39 Citations (Scopus)

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-κB/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.

Original languageEnglish (US)
Pages (from-to)313-322
Number of pages10
JournalMethods
Volume61
Issue number3
DOIs
StatePublished - Jun 15 2013

Fingerprint

Isotopes
Mass spectrometry
Assays
Mass Spectrometry
Dilution
Monitoring
Post Translational Protein Processing
Transcription Factor 3
Innate Immunity
Transcription Factor RelA
Proteins
Housekeeping
Workflow
Interferons
Peptides
Kinetics

Keywords

  • Innate immune response
  • Interferon response factor (IRF)
  • Nuclear factor-κB (NF-κB)
  • Post-translational modification
  • Selected reaction monitoring
  • Stable isotope dilution

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways. / Zhao, Yingxin; Brasier, Allan R.

In: Methods, Vol. 61, No. 3, 15.06.2013, p. 313-322.

Research output: Contribution to journalArticle

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