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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.

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Skyline display from replicate peak areas and imported targetedfull MS and MS/MS data from a high (2.0 fmol ng–1 BSA), medium (0.2 fmol ng–1 BSA), and low (0.02fmol ng–1 BSA) concentration of a phosphorylatedpeptide (DRVpYIHPF) spiked into BSA. Samples wererun on a Thermo Fisher LTQ-Velos, low-resolution instrument. The precursorion, in blue, is filtered from MS1 scans taken at the beginningof each cycle. At lower concentrations, interference becomes an issuefor the precursor in the MS1 scans; however, the filteredproduct ions from the targeted MS/MS remain selective and free frominterference producing a clear chromatographic peak. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the fragment ion (tandem MS) traces.
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fig2: Skyline display from replicate peak areas and imported targetedfull MS and MS/MS data from a high (2.0 fmol ng–1 BSA), medium (0.2 fmol ng–1 BSA), and low (0.02fmol ng–1 BSA) concentration of a phosphorylatedpeptide (DRVpYIHPF) spiked into BSA. Samples wererun on a Thermo Fisher LTQ-Velos, low-resolution instrument. The precursorion, in blue, is filtered from MS1 scans taken at the beginningof each cycle. At lower concentrations, interference becomes an issuefor the precursor in the MS1 scans; however, the filteredproduct ions from the targeted MS/MS remain selective and free frominterference producing a clear chromatographic peak. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the fragment ion (tandem MS) 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 imported targetedfull MS and MS/MS data from a high (2.0 fmol ng–1 BSA), medium (0.2 fmol ng–1 BSA), and low (0.02fmol ng–1 BSA) concentration of a phosphorylatedpeptide (DRVpYIHPF) spiked into BSA. Samples wererun on a Thermo Fisher LTQ-Velos, low-resolution instrument. The precursorion, in blue, is filtered from MS1 scans taken at the beginningof each cycle. At lower concentrations, interference becomes an issuefor the precursor in the MS1 scans; however, the filteredproduct ions from the targeted MS/MS remain selective and free frominterference producing a clear chromatographic peak. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the fragment ion (tandem MS) traces.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Skyline display from replicate peak areas and imported targetedfull MS and MS/MS data from a high (2.0 fmol ng–1 BSA), medium (0.2 fmol ng–1 BSA), and low (0.02fmol ng–1 BSA) concentration of a phosphorylatedpeptide (DRVpYIHPF) spiked into BSA. Samples wererun on a Thermo Fisher LTQ-Velos, low-resolution instrument. The precursorion, in blue, is filtered from MS1 scans taken at the beginningof each cycle. At lower concentrations, interference becomes an issuefor the precursor in the MS1 scans; however, the filteredproduct ions from the targeted MS/MS remain selective and free frominterference producing a clear chromatographic peak. Replicate peakareas show the reproducibility of the peaks and their compositionfrom the fragment ion (tandem MS) 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