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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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(A) Illustration of the immunoprecipitation(IP) method for EGFRand treatment groups utilized in these experiments. After IP, theEGFR corresponding band on SDS PAGE gel was excised and targeted analysisperformed on an LTQ Velos mass spectrometer. (B) Representation ofthe analytical approach for normalizing six EGFR phosphorylated peptidesto internal reference peptides (IRPs). IRPs are unmodified peptideswithin the protein of interest, in these experiments, EGFR.
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fig4: (A) Illustration of the immunoprecipitation(IP) method for EGFRand treatment groups utilized in these experiments. After IP, theEGFR corresponding band on SDS PAGE gel was excised and targeted analysisperformed on an LTQ Velos mass spectrometer. (B) Representation ofthe analytical approach for normalizing six EGFR phosphorylated peptidesto internal reference peptides (IRPs). IRPs are unmodified peptideswithin the protein of interest, in these experiments, EGFR.

Mentions: For quantitativeLC–MS/MS experiments, EGFR was immunoprecipitatedfrom the A431 cells with cetuximab in the presence of protease andphosphatase inhibitors, separated on an SDS-PAGE gel, and the EGFRband was excised and digested in-gel with trypsin (Figure 4). Modified peptides correspondingto phosphorylation at sites Y998 (MHLPSPTDSNFpYR),Y1110 (RPAGSVQNPVpYHNQPLNPAPSR), Y1172 (GSHQISLDNPDpYQQDFFPK), Y1197 (GSTAENAEpYLR), S991(MHLPpSPTDSNFYR), and S1166 (GSHQIpSLDNPDYQQDFFPK) within EGFR were targeted for quantification usingpSRM. Figure 6 shows the extracted ion chromatogramsusing Skyline for Y1197 for each treatment type. Phosphoserine sites(S991 and S1166) also were monitored using MS3 of the 98Da neutral loss ion corresponding to the neutral loss of phosphoricacid. Stable isotope-labeled peptides corresponding to sites Y998,Y1110, Y1172, and Y1197 were spiked in prior to pSRM analysis to permitcomparison of the IRP and SID methods for quantitation. Five unmodifiedpeptides from EGFR were selected for normalization using the IRP method.The location and domain position (extracellular, juxtamembrane, tyrosinekinase or cytoplasmic) of each reference and phosphorylated peptidein EGFR is presented in Figure S11 (SupportingInformation). Internal reference peptides were selected basedon high signal stability and a wide range of elution times (see FigureS12, Supporting Information). For eachtargeted peptide monitored, XICs were selected for three to five transitionsthat indicate modification specificity (when possible) and that generatedstrong pSRM transitions signals. Although modification site-specifictransitions do not always produce sufficient signal for quantitation,they frequently can be used to verify the site of modification. Inthe case of Y998 vs S991 phosphorylation, we were able to verify thesite not only with modification site-specific fragment ions but alsowith the comparison to the Y998 phosphorylated synthetic peptide.Extracted ion chromatograms for peptides Y998 and S991 (MHLPSPTDSNFpYR and MHLPpSPTDSNFYR, respectively; shownin Figure S2, Supporting Information) demonstratebaseline separation of these species under our experimental conditions.Since only one or two modification site-specific transitions weredetected, these ions were used primarily to distinguish the two distinctphosphorylated peptide signals. We utilized a few nonspecific transitionsfor quantitation since baseline separation was achieved. It is possiblethat the digestion of this peptide may be hindered when the tyrosineis phosphorylated (given its proximity to the tryptic cleavage site);we assumed that the relative amounts of both peptides (MHLPSPTDSNFpYR and MHLPpSPTDSNFYR) with respect totreatment should remain unaffected and digestion should be reproducible,thus the conclusion is the same. We believe that this assumption isreasonable since we are not comparing the absolute amount of thesetwo peptides and our data was consistent among the three biologicalreplicates. The absolute amount of the Y998 peptide estimated by SID,however, may be affected by this potential for missed cleavage. Thepeptide sequences, precursor m/z, and specific transitions are listed in Table S13, Supporting Information. Peak areas were calculated from thesum of transitions for the phosphopeptides and then divided by thepeak areas for EGFR internal reference peptides (for IRP) or its stableisotope-labeled standards (for SID). Three biological replicates wereperformed for each treatment group and three LC–pSRM–MSinjections for each sample.


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)

(A) Illustration of the immunoprecipitation(IP) method for EGFRand treatment groups utilized in these experiments. After IP, theEGFR corresponding band on SDS PAGE gel was excised and targeted analysisperformed on an LTQ Velos mass spectrometer. (B) Representation ofthe analytical approach for normalizing six EGFR phosphorylated peptidesto internal reference peptides (IRPs). IRPs are unmodified peptideswithin the protein of interest, in these experiments, EGFR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: (A) Illustration of the immunoprecipitation(IP) method for EGFRand treatment groups utilized in these experiments. After IP, theEGFR corresponding band on SDS PAGE gel was excised and targeted analysisperformed on an LTQ Velos mass spectrometer. (B) Representation ofthe analytical approach for normalizing six EGFR phosphorylated peptidesto internal reference peptides (IRPs). IRPs are unmodified peptideswithin the protein of interest, in these experiments, EGFR.
Mentions: For quantitativeLC–MS/MS experiments, EGFR was immunoprecipitatedfrom the A431 cells with cetuximab in the presence of protease andphosphatase inhibitors, separated on an SDS-PAGE gel, and the EGFRband was excised and digested in-gel with trypsin (Figure 4). Modified peptides correspondingto phosphorylation at sites Y998 (MHLPSPTDSNFpYR),Y1110 (RPAGSVQNPVpYHNQPLNPAPSR), Y1172 (GSHQISLDNPDpYQQDFFPK), Y1197 (GSTAENAEpYLR), S991(MHLPpSPTDSNFYR), and S1166 (GSHQIpSLDNPDYQQDFFPK) within EGFR were targeted for quantification usingpSRM. Figure 6 shows the extracted ion chromatogramsusing Skyline for Y1197 for each treatment type. Phosphoserine sites(S991 and S1166) also were monitored using MS3 of the 98Da neutral loss ion corresponding to the neutral loss of phosphoricacid. Stable isotope-labeled peptides corresponding to sites Y998,Y1110, Y1172, and Y1197 were spiked in prior to pSRM analysis to permitcomparison of the IRP and SID methods for quantitation. Five unmodifiedpeptides from EGFR were selected for normalization using the IRP method.The location and domain position (extracellular, juxtamembrane, tyrosinekinase or cytoplasmic) of each reference and phosphorylated peptidein EGFR is presented in Figure S11 (SupportingInformation). Internal reference peptides were selected basedon high signal stability and a wide range of elution times (see FigureS12, Supporting Information). For eachtargeted peptide monitored, XICs were selected for three to five transitionsthat indicate modification specificity (when possible) and that generatedstrong pSRM transitions signals. Although modification site-specifictransitions do not always produce sufficient signal for quantitation,they frequently can be used to verify the site of modification. Inthe case of Y998 vs S991 phosphorylation, we were able to verify thesite not only with modification site-specific fragment ions but alsowith the comparison to the Y998 phosphorylated synthetic peptide.Extracted ion chromatograms for peptides Y998 and S991 (MHLPSPTDSNFpYR and MHLPpSPTDSNFYR, respectively; shownin Figure S2, Supporting Information) demonstratebaseline separation of these species under our experimental conditions.Since only one or two modification site-specific transitions weredetected, these ions were used primarily to distinguish the two distinctphosphorylated peptide signals. We utilized a few nonspecific transitionsfor quantitation since baseline separation was achieved. It is possiblethat the digestion of this peptide may be hindered when the tyrosineis phosphorylated (given its proximity to the tryptic cleavage site);we assumed that the relative amounts of both peptides (MHLPSPTDSNFpYR and MHLPpSPTDSNFYR) with respect totreatment should remain unaffected and digestion should be reproducible,thus the conclusion is the same. We believe that this assumption isreasonable since we are not comparing the absolute amount of thesetwo peptides and our data was consistent among the three biologicalreplicates. The absolute amount of the Y998 peptide estimated by SID,however, may be affected by this potential for missed cleavage. Thepeptide sequences, precursor m/z, and specific transitions are listed in Table S13, Supporting Information. Peak areas were calculated from thesum of transitions for the phosphopeptides and then divided by thepeak areas for EGFR internal reference peptides (for IRP) or its stableisotope-labeled standards (for SID). Three biological replicates wereperformed for each treatment group and three LC–pSRM–MSinjections for each sample.

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