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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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Internal reference peptide proof-of-principle experiment. Syntheticphosphopeptides were spiked into a standard bovine serum albumin (BSA)protein digest at increasing concentrations (0.064–12.8 fmolμL–1). Phosphorylated peptides were targetedemploying pSRM using a linear ion-trap mass spectrometer. Data isnormalized by dividing targeted phosphorylated peptide peak area (sumof three to four transitions) by BSA peptide peak areas (sum of threetransitions).
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fig1: Internal reference peptide proof-of-principle experiment. Syntheticphosphopeptides were spiked into a standard bovine serum albumin (BSA)protein digest at increasing concentrations (0.064–12.8 fmolμL–1). Phosphorylated peptides were targetedemploying pSRM using a linear ion-trap mass spectrometer. Data isnormalized by dividing targeted phosphorylated peptide peak area (sumof three to four transitions) by BSA peptide peak areas (sum of threetransitions).

Mentions: To test the ability of the IRP method to detect differences in modificationstoichiometry as changes in normalized pSRM ratio, we performed proof-of-principleexperiments by spiking synthetic phosphopeptides into a BSA background(see Figure 1). The peptides, DRVpYIHPF (angiotensin II) and IKNLQpSLDPSH (cholecystokinin10–20), were spiked into 100 μL of 6 μg mL–1 BSA digest at 0.01, 0.02, 0.05, 0.10, 0.20, 0.50,1.0, and 2.0 fmol ng–1 BSA. These concentrationswere chosen to mimic the low abundance phosphorylation events thatoccur in biological systems;12 these spikeconcentrations correlated to 0.12–14% stoichiometry relativeto BSA.


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)

Internal reference peptide proof-of-principle experiment. Syntheticphosphopeptides were spiked into a standard bovine serum albumin (BSA)protein digest at increasing concentrations (0.064–12.8 fmolμL–1). Phosphorylated peptides were targetedemploying pSRM using a linear ion-trap mass spectrometer. Data isnormalized by dividing targeted phosphorylated peptide peak area (sumof three to four transitions) by BSA peptide peak areas (sum of threetransitions).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Internal reference peptide proof-of-principle experiment. Syntheticphosphopeptides were spiked into a standard bovine serum albumin (BSA)protein digest at increasing concentrations (0.064–12.8 fmolμL–1). Phosphorylated peptides were targetedemploying pSRM using a linear ion-trap mass spectrometer. Data isnormalized by dividing targeted phosphorylated peptide peak area (sumof three to four transitions) by BSA peptide peak areas (sum of threetransitions).
Mentions: To test the ability of the IRP method to detect differences in modificationstoichiometry as changes in normalized pSRM ratio, we performed proof-of-principleexperiments by spiking synthetic phosphopeptides into a BSA background(see Figure 1). The peptides, DRVpYIHPF (angiotensin II) and IKNLQpSLDPSH (cholecystokinin10–20), were spiked into 100 μL of 6 μg mL–1 BSA digest at 0.01, 0.02, 0.05, 0.10, 0.20, 0.50,1.0, and 2.0 fmol ng–1 BSA. These concentrationswere chosen to mimic the low abundance phosphorylation events thatoccur in biological systems;12 these spikeconcentrations correlated to 0.12–14% stoichiometry relativeto BSA.

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