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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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Median CV across technical replicates plotted againstamount ofspiked in phosphorylated peptide standards. Data point colors correspondto reference peptide used for normalization. The median CVs decreaseas the amount of phosphopeptide spiked in background (BSA digest)increases.
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fig3: Median CV across technical replicates plotted againstamount ofspiked in phosphorylated peptide standards. Data point colors correspondto reference peptide used for normalization. The median CVs decreaseas the amount of phosphopeptide spiked in background (BSA digest)increases.

Mentions: MS/MS and MS3 data from five replicateLC–MSanalyses of BSA digests spiked with both phosphopeptides were normalizedby the IRP method and concentration response curves are shown in FigureS3 (Supporting Information). Concentration–responsecurve slopes, correlation coefficients (r2), and coefficients of variation (CV) for all reference peptidesare presented in Table 1. Individual plotsfor all of the measured peptides are shown in Figures S5–S7(Supporting Information) and plots of themedian CVs for all replicates are in Figure 3. Despite the fact that all of the normalization peptides were derivedfrom an equal amount of BSA in the sample, values for r2, slope, and CV for normalization varied. CV values rangedfrom 7.3 to 15.7% (median 10%). The the utilization of the BSA referencepeptide YICDNQDTISSK for quantation of the phosphorylated peptidesyielded the highest r2 values (≥0.96),lowest slope, and lowest median CV (≤10.8%) compared to theother reference peptides. Similar values for r2, slope, and CV were obtained for both MS/MS and MS3 measurements. The highest median CV was observed at the lowest phosphopeptidespike amount (0.128 fmol) for both phosphopeptides (MS/MS and MS3 data; Figure 3, Supporting Information). Plots derived using single BSA reference peptides and using thesum of all BSA reference peptides are presented in Figures S8–S10(Supporting Information). These experimentswere performed on a linear ion trap with automatic gain control (AGC),which limits filling of the trap at higher ion currents, thus potentiallylimiting linear dynamic range. In these studies, IRP-normalized signalsappeared linear over the 200-fold concentration range examined, whichsuggests that AGC has little impact on response under the conditionsof our analyses.


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)

Median CV across technical replicates plotted againstamount ofspiked in phosphorylated peptide standards. Data point colors correspondto reference peptide used for normalization. The median CVs decreaseas the amount of phosphopeptide spiked in background (BSA digest)increases.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Median CV across technical replicates plotted againstamount ofspiked in phosphorylated peptide standards. Data point colors correspondto reference peptide used for normalization. The median CVs decreaseas the amount of phosphopeptide spiked in background (BSA digest)increases.
Mentions: MS/MS and MS3 data from five replicateLC–MSanalyses of BSA digests spiked with both phosphopeptides were normalizedby the IRP method and concentration response curves are shown in FigureS3 (Supporting Information). Concentration–responsecurve slopes, correlation coefficients (r2), and coefficients of variation (CV) for all reference peptidesare presented in Table 1. Individual plotsfor all of the measured peptides are shown in Figures S5–S7(Supporting Information) and plots of themedian CVs for all replicates are in Figure 3. Despite the fact that all of the normalization peptides were derivedfrom an equal amount of BSA in the sample, values for r2, slope, and CV for normalization varied. CV values rangedfrom 7.3 to 15.7% (median 10%). The the utilization of the BSA referencepeptide YICDNQDTISSK for quantation of the phosphorylated peptidesyielded the highest r2 values (≥0.96),lowest slope, and lowest median CV (≤10.8%) compared to theother reference peptides. Similar values for r2, slope, and CV were obtained for both MS/MS and MS3 measurements. The highest median CV was observed at the lowest phosphopeptidespike amount (0.128 fmol) for both phosphopeptides (MS/MS and MS3 data; Figure 3, Supporting Information). Plots derived using single BSA reference peptides and using thesum of all BSA reference peptides are presented in Figures S8–S10(Supporting Information). These experimentswere performed on a linear ion trap with automatic gain control (AGC),which limits filling of the trap at higher ion currents, thus potentiallylimiting linear dynamic range. In these studies, IRP-normalized signalsappeared linear over the 200-fold concentration range examined, whichsuggests that AGC has little impact on response under the conditionsof our analyses.

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