title: 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-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.
authors: Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors: Yingxin
authors: Y
authors: Zhao
authors: Allan R
authors: AR
authors: Brasier
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay; D002247:Carbon Isotopes; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007201:Indicator Dilution Techniques; D050838:Interferon Regulatory Factor-3 / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000032:analysis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods; D051996:Transcription Factor RelA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D050838:Interferon Regulatory Factor-3; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; C498143:RELA protein, human; D051996:Transcription Factor RelA