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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
A431 cells untreated, modulated with EGF, or cotreatedwith inhibitor(cetuximab or gefitinib) followed by EGF exhibit different EGFR activationstatuses. Immunoblot showing EGFR activation prior to and after treatment(s)(phosphorylation at Y998 and Y1172). Both phosphorylated forms ofthe receptor were targeted in the pSRM-MS method. Input lane is 5%of total protein load, and IP lane is post-immunoprecipitation. Controllanes show the IP performed using mouse IgG. All treated cells wereserum-starved overnight prior to any treatment.
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fig5: A431 cells untreated, modulated with EGF, or cotreatedwith inhibitor(cetuximab or gefitinib) followed by EGF exhibit different EGFR activationstatuses. Immunoblot showing EGFR activation prior to and after treatment(s)(phosphorylation at Y998 and Y1172). Both phosphorylated forms ofthe receptor were targeted in the pSRM-MS method. Input lane is 5%of total protein load, and IP lane is post-immunoprecipitation. Controllanes show the IP performed using mouse IgG. All treated cells wereserum-starved overnight prior to any treatment.

Mentions: Immunoblot analyses were performedpre- and post IP for each treatment group for total EGFR and specific pY sites Y998 and Y1172. EGFR specific pY sites Y998 andY1172 were chosen for immunoblot analysis because both sites havehigh-quality, commercially available site-specific antibodies. Phosphorylationat both residues has been linked to pertinent EGFR biology with Y998representing a phosphorylation site implicated in receptor endocytosisand Y1172 representing a site of autophosphorylation. The purposeof these studies was not to produce equal levels of inhibition butto produce detectable differences in phosphorylation between stimulatedand inhibited states using at least two known inhibitors of EGFR.The amount of receptor phosphorylation at sites Y998 and Y1172 inA431 cells (Figure 5) was significantly elevatedin EGF treated samples over proliferating controls. As expected, bothcetuximab and gefitinib inhibitor treatments decreased the amountof tyrosine phosphorylation detected when compared to EGF treatmentalone. The EGFR tyrosine kinase inhibitor gefitinib at 500 nM decreasedtyrosine phosphorylation below basal levels at both sites, whereasat a dose of 10 μg mL–1, cetuximab was notas effective at inhibiting EGFR phosphorylation.


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)

A431 cells untreated, modulated with EGF, or cotreatedwith inhibitor(cetuximab or gefitinib) followed by EGF exhibit different EGFR activationstatuses. Immunoblot showing EGFR activation prior to and after treatment(s)(phosphorylation at Y998 and Y1172). Both phosphorylated forms ofthe receptor were targeted in the pSRM-MS method. Input lane is 5%of total protein load, and IP lane is post-immunoprecipitation. Controllanes show the IP performed using mouse IgG. All treated cells wereserum-starved overnight prior to any treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: A431 cells untreated, modulated with EGF, or cotreatedwith inhibitor(cetuximab or gefitinib) followed by EGF exhibit different EGFR activationstatuses. Immunoblot showing EGFR activation prior to and after treatment(s)(phosphorylation at Y998 and Y1172). Both phosphorylated forms ofthe receptor were targeted in the pSRM-MS method. Input lane is 5%of total protein load, and IP lane is post-immunoprecipitation. Controllanes show the IP performed using mouse IgG. All treated cells wereserum-starved overnight prior to any treatment.
Mentions: Immunoblot analyses were performedpre- and post IP for each treatment group for total EGFR and specific pY sites Y998 and Y1172. EGFR specific pY sites Y998 andY1172 were chosen for immunoblot analysis because both sites havehigh-quality, commercially available site-specific antibodies. Phosphorylationat both residues has been linked to pertinent EGFR biology with Y998representing a phosphorylation site implicated in receptor endocytosisand Y1172 representing a site of autophosphorylation. The purposeof these studies was not to produce equal levels of inhibition butto produce detectable differences in phosphorylation between stimulatedand inhibited states using at least two known inhibitors of EGFR.The amount of receptor phosphorylation at sites Y998 and Y1172 inA431 cells (Figure 5) was significantly elevatedin EGF treated samples over proliferating controls. As expected, bothcetuximab and gefitinib inhibitor treatments decreased the amountof tyrosine phosphorylation detected when compared to EGF treatmentalone. The EGFR tyrosine kinase inhibitor gefitinib at 500 nM decreasedtyrosine phosphorylation below basal levels at both sites, whereasat a dose of 10 μg mL–1, cetuximab was notas effective at inhibiting EGFR phosphorylation.

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