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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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Treatment groups showthe same trends when pY peptidesare normalized to an internal reference peptide or its stable isotopelabeled counterpart. EGFR peptide pY1172 (GSHQISLDNPDpYQQDFFPK) normalized to an (A) internal reference peptide(IPLENLQIIR) and (B) its pY SID peptide standard.(C) Phosphorylation index for each EGFR pY peptideand cell treatment group. Similar trends are observed for each pY targeted peptide after normalization to an IRP (red)or its stable isotope labeled counterpart (purple). The phosphorylationindex is normalized to EGF (100%) stimulated cells. These data representthree technical LC–pSRM–MS injections of biologicalreplicate 1. ND, not detected in LC–pSRM–MS experiments.The underlined amino acid indicates which amino acid was stable isotope-labeled.For biological replicate 1, CV values ranged between 9.8–47%and 7.3–32% for the IRP and SID method, respectively. The higherCV values were generated from Geftinib + EGF samples, and the medianCV values ranged were 21% and 24%, respectively.
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fig7: Treatment groups showthe same trends when pY peptidesare normalized to an internal reference peptide or its stable isotopelabeled counterpart. EGFR peptide pY1172 (GSHQISLDNPDpYQQDFFPK) normalized to an (A) internal reference peptide(IPLENLQIIR) and (B) its pY SID peptide standard.(C) Phosphorylation index for each EGFR pY peptideand cell treatment group. Similar trends are observed for each pY targeted peptide after normalization to an IRP (red)or its stable isotope labeled counterpart (purple). The phosphorylationindex is normalized to EGF (100%) stimulated cells. These data representthree technical LC–pSRM–MS injections of biologicalreplicate 1. ND, not detected in LC–pSRM–MS experiments.The underlined amino acid indicates which amino acid was stable isotope-labeled.For biological replicate 1, CV values ranged between 9.8–47%and 7.3–32% for the IRP and SID method, respectively. The higherCV values were generated from Geftinib + EGF samples, and the medianCV values ranged were 21% and 24%, respectively.

Mentions: The data generated by analyses of immunoprecipitatedEGFR frombiological experiment 1 by the IRP and SID methods are shown in Figure 7. Both methods yielded similar measures of phosphorylationat Y1172 (GSHQISLDNPDpYQQDFFPK), its stimulationby EGF, and inhibition by cetuximab and gefitinib (Figure 7A,B). The IRP method produced similar results toSID for all four phosphotyrosine sites (Y998, Y1110, Y1172, and Y1197)(Figure 7C and Figures S24–S27, Supporting Information). These results are alsoconsistent with the immunoblot analysis for Y1172 shown in Figure 5. EGF-treated stimulation produced the highest normalizedpSRM signal, whereas samples cotreated with 500 nM gefitinib showedprofound decreases in Y1172 phosphorylation to below basal (proliferating)levels. Co-treatment with cetuximab produced less inhibition in Y1172phosphorylation to near basal levels. Figure 7C represents the degree of site-specific phosphorylation relativeto that for EGF stimulation by a phosphorylation index, which wascalculated as the ratio of the proliferating (P), gefitinib (G+E),or cetuximab (C+E) normalized pSRM signal to the EGF stimulated normalizedpSRM signal (eq 1). Although we monitored thecorresponding peptides without phosphorylation (data not shown), wedid not see any appreciable changes in the quantitation of these peptides,most likely due to the low stoichiometry of the phosphorylation (datanot shown).1


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)

Treatment groups showthe same trends when pY peptidesare normalized to an internal reference peptide or its stable isotopelabeled counterpart. EGFR peptide pY1172 (GSHQISLDNPDpYQQDFFPK) normalized to an (A) internal reference peptide(IPLENLQIIR) and (B) its pY SID peptide standard.(C) Phosphorylation index for each EGFR pY peptideand cell treatment group. Similar trends are observed for each pY targeted peptide after normalization to an IRP (red)or its stable isotope labeled counterpart (purple). The phosphorylationindex is normalized to EGF (100%) stimulated cells. These data representthree technical LC–pSRM–MS injections of biologicalreplicate 1. ND, not detected in LC–pSRM–MS experiments.The underlined amino acid indicates which amino acid was stable isotope-labeled.For biological replicate 1, CV values ranged between 9.8–47%and 7.3–32% for the IRP and SID method, respectively. The higherCV values were generated from Geftinib + EGF samples, and the medianCV values ranged were 21% and 24%, respectively.
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Related In: Results  -  Collection

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fig7: Treatment groups showthe same trends when pY peptidesare normalized to an internal reference peptide or its stable isotopelabeled counterpart. EGFR peptide pY1172 (GSHQISLDNPDpYQQDFFPK) normalized to an (A) internal reference peptide(IPLENLQIIR) and (B) its pY SID peptide standard.(C) Phosphorylation index for each EGFR pY peptideand cell treatment group. Similar trends are observed for each pY targeted peptide after normalization to an IRP (red)or its stable isotope labeled counterpart (purple). The phosphorylationindex is normalized to EGF (100%) stimulated cells. These data representthree technical LC–pSRM–MS injections of biologicalreplicate 1. ND, not detected in LC–pSRM–MS experiments.The underlined amino acid indicates which amino acid was stable isotope-labeled.For biological replicate 1, CV values ranged between 9.8–47%and 7.3–32% for the IRP and SID method, respectively. The higherCV values were generated from Geftinib + EGF samples, and the medianCV values ranged were 21% and 24%, respectively.
Mentions: The data generated by analyses of immunoprecipitatedEGFR frombiological experiment 1 by the IRP and SID methods are shown in Figure 7. Both methods yielded similar measures of phosphorylationat Y1172 (GSHQISLDNPDpYQQDFFPK), its stimulationby EGF, and inhibition by cetuximab and gefitinib (Figure 7A,B). The IRP method produced similar results toSID for all four phosphotyrosine sites (Y998, Y1110, Y1172, and Y1197)(Figure 7C and Figures S24–S27, Supporting Information). These results are alsoconsistent with the immunoblot analysis for Y1172 shown in Figure 5. EGF-treated stimulation produced the highest normalizedpSRM signal, whereas samples cotreated with 500 nM gefitinib showedprofound decreases in Y1172 phosphorylation to below basal (proliferating)levels. Co-treatment with cetuximab produced less inhibition in Y1172phosphorylation to near basal levels. Figure 7C represents the degree of site-specific phosphorylation relativeto that for EGF stimulation by a phosphorylation index, which wascalculated as the ratio of the proliferating (P), gefitinib (G+E),or cetuximab (C+E) normalized pSRM signal to the EGF stimulated normalizedpSRM signal (eq 1). Although we monitored thecorresponding peptides without phosphorylation (data not shown), wedid not see any appreciable changes in the quantitation of these peptides,most likely due to the low stoichiometry of the phosphorylation (datanot shown).1

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