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

Sherrod SD, Myers MV, Li M, Myers JS, Carpenter KL, Maclean B, Maccoss MJ, Liebler DC, Ham AJ - J. Proteome Res. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Jim Ayers Institute of Precancer Detection and Diagnosis and §Department of Biostatistics, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States.

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.

Show MeSH
Skyline display from replicate peak areas and importedtargetedMS/MS data to produce pSRM traces for GSTAENAEpYLRfrom A431 cells not treated (proliferating), modulated with EGF, orcotreated with inhibitor (cetuximab or gefitinib) followed by EGF.Samples were acquired using a Thermo Fisher LTQ-Velos. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the pSRM traces.
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fig6: Skyline display from replicate peak areas and importedtargetedMS/MS data to produce pSRM traces for GSTAENAEpYLRfrom A431 cells not treated (proliferating), modulated with EGF, orcotreated with inhibitor (cetuximab or gefitinib) followed by EGF.Samples were acquired using a Thermo Fisher LTQ-Velos. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the pSRM traces.

Mentions: Full support for pSRM usingchromatograms extracted from targeted MS/MS spectra for peak areacalculations was implemented in the Skyline software tool, as shownin Figures 2, 6 andS1 (Supporting Information), and releasedin version 1.1. These new features included method export for ThermoFisherLTQ instruments as well as chromatogram extraction from MS/MS spectraat targeted product ion mass-to-charge ratios, making available forpSRM many existing Skyline features proven in SRM experiments withtriple quadrupole mass spectrometers.


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

Sherrod SD, Myers MV, Li M, Myers JS, Carpenter KL, Maclean B, Maccoss MJ, Liebler DC, Ham AJ - J. Proteome Res. (2012)

Skyline display from replicate peak areas and importedtargetedMS/MS data to produce pSRM traces for GSTAENAEpYLRfrom A431 cells not treated (proliferating), modulated with EGF, orcotreated with inhibitor (cetuximab or gefitinib) followed by EGF.Samples were acquired using a Thermo Fisher LTQ-Velos. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the pSRM traces.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368409&req=5

fig6: Skyline display from replicate peak areas and importedtargetedMS/MS data to produce pSRM traces for GSTAENAEpYLRfrom A431 cells not treated (proliferating), modulated with EGF, orcotreated with inhibitor (cetuximab or gefitinib) followed by EGF.Samples were acquired using a Thermo Fisher LTQ-Velos. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the pSRM traces.
Mentions: Full support for pSRM usingchromatograms extracted from targeted MS/MS spectra for peak areacalculations was implemented in the Skyline software tool, as shownin Figures 2, 6 andS1 (Supporting Information), and releasedin version 1.1. These new features included method export for ThermoFisherLTQ instruments as well as chromatogram extraction from MS/MS spectraat targeted product ion mass-to-charge ratios, making available forpSRM many existing Skyline features proven in SRM experiments withtriple quadrupole mass spectrometers.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Jim Ayers Institute of Precancer Detection and Diagnosis and §Department of Biostatistics, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States.

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.

Show MeSH