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Label-free quantitation of protein modifications by pseudo selected reaction monitoring with internal reference peptides.

ABSTRACT: Liquid chromatography tandem mass spectrometry (LC-MS/MS) based methods provide powerful tools for the quantitative analysis of modified proteins. We have developed a label-free approach using internal reference peptides (IRP) from the target protein for signal normalization without the need for isotope labeling. Ion-trap mass spectrometry and pseudo-selected reaction monitoring (pSRM) were used to acquire full MS/MS and MS(3) spectra from target peptides. Skyline, a widely used software for SRM experiments, was used for chromatographic ion extraction. Phosphopeptides spiked into a BSA background yielded concentration response curves with high correlation coefficients (typically >0.9) and low coefficients of variation (?15%) over a 200-fold concentration range. Stable isotope dilution (SID) and IRP methods were compared for quantitation of six site-specific phosphorylations in the epidermal growth factor receptor (EGFR) in epidermal growth factor-stimulated A431 cells with or without the addition of EGFR inhibitors cetuximab and gefitinib. Equivalent responses were observed with both IRP and SID methods, although analyses using the IRP method typically had higher median CVs (22-31%) than SID (10-20%). Analyses using both methods were consistent with immunoblot using site-selective antibodies. The ease of implementation and the suitability for targeted quantitative comparisons make this method suitable for broad application in protein biochemistry.

SUBMITTER: Sherrod SD

PROVIDER: S-EPMC3368409 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Label-free quantitation of protein modifications by pseudo selected reaction monitoring with internal reference peptides.

Sherrod Stacy D SD Myers Matthew V MV Li Ming M Myers Jeremy S JS Carpenter Kristin L KL Maclean Brendan B Maccoss Michael J MJ Liebler Daniel C DC Ham Amy-Joan L AJ

Journal of proteome research 20120517 6

Liquid chromatography tandem mass spectrometry (LC-MS/MS) based methods provide powerful tools for the quantitative analysis of modified proteins. We have developed a label-free approach using internal reference peptides (IRP) from the target protein for signal normalization without the need for isotope labeling. Ion-trap mass spectrometry and pseudo-selected reaction monitoring (pSRM) were used to acquire full MS/MS and MS(3) spectra from target peptides. Skyline, a widely used software for SRM ...[more]

PMID: 22559222

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Project description:The innate immune response (IIR) is a coordinated intracellular signaling network activated by the presence of pathogen-associated molecular patterns that limits pathogen spread and induces adaptive immunity. Although the precise temporal activation of the various arms of the IIR is a critical factor in the outcome of a disease, currently there are no quantitative multiplex methods for its measurement. In this study, we investigate the temporal activation pattern of the IIR in response to intracellular double-stranded RNA stimulation using a quantitative 10-plex stable isotope dilution-selected reaction monitoring-MS assay. We were able to observe rapid activation of both NF-?B and IRF3 signaling arms, with IRF3 demonstrating a transient response, whereas NF-?B underwent a delayed secondary amplification phase. Our measurements of the NF-?B-I?B? negative feedback loop indicate that about 20% of I?B? in the unstimulated cell is located within the nucleus and represents a population that is rapidly degraded in response to double-stranded RNA. Later in the time course of stimulation, the nuclear I?B? pool is repopulated first prior to its cytoplasmic accumulation. Examination of the IRF3 pathway components shows that double-stranded RNA induces initial consumption of the RIG-I PRR and the IRF3 kinase (TBK1). Stable isotope dilution-selected reaction monitoring-MS measurements after siRNA-mediated IRF3 or RelA knockdown suggests that a low nuclear threshold of NF-?B is required for inducing target gene expression, and that there is cross-inhibition of the NF-?B and IRF3 signaling arms. Finally, we were able to measure delayed noncanonical NF-?B activation by quantifying the abundance of the processed (52 kDa) NF-?B2 subunit in the nucleus. We conclude that quantitative proteomics measurement of the individual signaling arms of the IIR in response to system perturbations is significantly enabled by stable isotope dilution-selected reaction monitoring-MS-based quantification, and that this technique will reveal novel insights into the dynamics and connectivity of the IIR.

Dataset Information

Label-free quantitation of protein modifications by pseudo selected reaction monitoring with internal reference peptides.

Publications

Label-free quantitation of protein modifications by pseudo selected reaction monitoring with internal reference peptides.

Similar Datasets

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