TY - JOUR
T1 - Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways
AU - Zhao, Yingxin
AU - Brasier, Allan R.
N1 - Funding Information:
Supported by Institute for Human Infections and Immunity Pilot Program, NIAID PO1 AI062885, NCATS UL1TR000071, NIAID Clinical Proteomics Center HHSN272200800048C, NHLBI Proteomics Center for Airway Inflammation NIH-NHLBI-HHSN268201000037C and NIEHS P30 ES006676.
PY - 2013/6/15
Y1 - 2013/6/15
N2 - 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.
AB - 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.
KW - Innate immune response
KW - Interferon response factor (IRF)
KW - Nuclear factor-κB (NF-κB)
KW - Post-translational modification
KW - Selected reaction monitoring
KW - Stable isotope dilution
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U2 - 10.1016/j.ymeth.2013.02.001
DO - 10.1016/j.ymeth.2013.02.001
M3 - Review article
C2 - 23410677
AN - SCOPUS:84879597199
SN - 1046-2023
VL - 61
SP - 313
EP - 322
JO - Methods
JF - Methods
IS - 3
ER -