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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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MS3 trendplots for two phosphoserine modified peptidesbased on treatment group. Phosphopeptide pS991 (MHLPpSPTDSNFYR) normalized to an (A) IRP or (B) pY SID peptide standard (MHLPSPTDSNFpYR) shows similar trends. These data are similar to trends observedfor pY peptide MHLPSPTDSNFpYR. Phosphopeptide pS1166 (GSHQIpSLDNPDYQQDFFPK) normalizedto an (C) IRP or (D) pY SID peptide standard (GSHQISLDNPDpYQQDFFPK) show similar trends. Thesedata show that MS3 measurements can be used for quantificationof protein modifications. These data represent three technical injectsof biological replicate 2. The underlined amino acid indicates whichamino acid was stable-isotope labeled.
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fig8: MS3 trendplots for two phosphoserine modified peptidesbased on treatment group. Phosphopeptide pS991 (MHLPpSPTDSNFYR) normalized to an (A) IRP or (B) pY SID peptide standard (MHLPSPTDSNFpYR) shows similar trends. These data are similar to trends observedfor pY peptide MHLPSPTDSNFpYR. Phosphopeptide pS1166 (GSHQIpSLDNPDYQQDFFPK) normalizedto an (C) IRP or (D) pY SID peptide standard (GSHQISLDNPDpYQQDFFPK) show similar trends. Thesedata show that MS3 measurements can be used for quantificationof protein modifications. These data represent three technical injectsof biological replicate 2. The underlined amino acid indicates whichamino acid was stable-isotope labeled.

Mentions: For pS and pT peptides whose MS/MS spectra are dominated by neutral loss of H3PO4, measurements based on MS3 fragmentationof the neutral loss ion may offer higher confidence sequence-specificdetection. MS3 measurements for relative quantificationof phosphoserine modifications were performed on peptides for EGFRsites S991 (MHLPpSPTDSNFYR) [M + 2H – H3PO4]2+ and S1166 (GSHQIpSLDNPDYQQDFFPK) [M + 3H – H3PO4]3+. Peak areas from MS3 measurements were normalizedto MS/MS-derived peak areas for the 5 EGFR IRP sequences describedabove, as well as to the MS/MS-derived peak areas for the isotope-labeled pY peptide standards used for SID analyses of the pY forms of these sequences (see above). Median CV plotsusing the IRP method for S991 and S1166 phosphopeptides (Figures S18and S19, Supporting Information) indicatethat normalization to the IPLENLQIIR peptide produced the smallestmeasurement variation, which was comparable to that achieved withnormalization to the synthetic pY SID peptide sequenceanalogs. Both analysis methods yielded similar estimates of phosphorylationchanges induced by EGF and the effects of cetuximab and gefitinib(Figure 8 and Figures S28 and S29, Supporting Information).


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)

MS3 trendplots for two phosphoserine modified peptidesbased on treatment group. Phosphopeptide pS991 (MHLPpSPTDSNFYR) normalized to an (A) IRP or (B) pY SID peptide standard (MHLPSPTDSNFpYR) shows similar trends. These data are similar to trends observedfor pY peptide MHLPSPTDSNFpYR. Phosphopeptide pS1166 (GSHQIpSLDNPDYQQDFFPK) normalizedto an (C) IRP or (D) pY SID peptide standard (GSHQISLDNPDpYQQDFFPK) show similar trends. Thesedata show that MS3 measurements can be used for quantificationof protein modifications. These data represent three technical injectsof biological replicate 2. The underlined amino acid indicates whichamino acid was stable-isotope labeled.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: MS3 trendplots for two phosphoserine modified peptidesbased on treatment group. Phosphopeptide pS991 (MHLPpSPTDSNFYR) normalized to an (A) IRP or (B) pY SID peptide standard (MHLPSPTDSNFpYR) shows similar trends. These data are similar to trends observedfor pY peptide MHLPSPTDSNFpYR. Phosphopeptide pS1166 (GSHQIpSLDNPDYQQDFFPK) normalizedto an (C) IRP or (D) pY SID peptide standard (GSHQISLDNPDpYQQDFFPK) show similar trends. Thesedata show that MS3 measurements can be used for quantificationof protein modifications. These data represent three technical injectsof biological replicate 2. The underlined amino acid indicates whichamino acid was stable-isotope labeled.
Mentions: For pS and pT peptides whose MS/MS spectra are dominated by neutral loss of H3PO4, measurements based on MS3 fragmentationof the neutral loss ion may offer higher confidence sequence-specificdetection. MS3 measurements for relative quantificationof phosphoserine modifications were performed on peptides for EGFRsites S991 (MHLPpSPTDSNFYR) [M + 2H – H3PO4]2+ and S1166 (GSHQIpSLDNPDYQQDFFPK) [M + 3H – H3PO4]3+. Peak areas from MS3 measurements were normalizedto MS/MS-derived peak areas for the 5 EGFR IRP sequences describedabove, as well as to the MS/MS-derived peak areas for the isotope-labeled pY peptide standards used for SID analyses of the pY forms of these sequences (see above). Median CV plotsusing the IRP method for S991 and S1166 phosphopeptides (Figures S18and S19, Supporting Information) indicatethat normalization to the IPLENLQIIR peptide produced the smallestmeasurement variation, which was comparable to that achieved withnormalization to the synthetic pY SID peptide sequenceanalogs. Both analysis methods yielded similar estimates of phosphorylationchanges induced by EGF and the effects of cetuximab and gefitinib(Figure 8 and Figures S28 and S29, Supporting Information).

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