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 isotopic 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.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling

“…The SID-SRM-MS assays were developed as described previously (24,25). For each targeted protein, two or three peptides were initially selected and then the sensitivity and selectivity of these were experimentally evaluated as described previously (24,25 lysine to a 99% isotopic enrichment (Thermo Scientific).…”

“…For each targeted protein, two or three peptides were initially selected and then the sensitivity and selectivity of these were experimentally evaluated as described previously (24,25 lysine to a 99% isotopic enrichment (Thermo Scientific). The amount of stable isotope standard (SIS) peptides was determined by amino acid analysis.…”

CDK9-Dependent Transcriptional Elongation in the Innate Interferon-Stimulated Gene Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells

“…The SID-SRM-MS assays of CDK9, BRD4, IRF3, IRF1, IRF7, STAT1, RSV N protein, and RSV NS1 were developed using a workflow described in previous publications (45,46). The signature peptides and SRM parameters are listed in lysine to a 99% isotopic enrichment (Thermo Scientific).…”

“…The proteins immunoprecipitated with anti-CDK9 antibody were captured by protein A magnetic beads (Dynal Inc.). The proteins were trypsin digested on the beads as described previously (45,46). Briefly, the beads were washed with PBS three times and then resuspended in 30 l of 50 mM ammonium hydrogen carbonate (pH 7.8), and 20 l of a 0.1-g/l solution of trypsin was added.…”

Proteomic Approaches and Identification of Novel Therapeutic Targets for Alcoholism

“…A better characterization of PTMs will be achieved through the development of more sensitive, refined, and improved methodologies and analytical tools. Indeed, modification-specific proteomics, which enables selective enrichment, purification, separation, and identification of various PTMs, will allow increasingly detailed analysis and quantitation of PTMs within proteins (Chung et al, 2013;Engholm-Keller and Larsen, 2013;Jensen, 2000;Zhao and Brasier, 2013) and will help elucidate their role in complex biological diseases like alcoholism. A reduction in the quantity of material required and analysis time necessary to obtain relevant proteomics data, while maintaining rigorous quality control, is also desirable.…”