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Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides
[Image: see text] 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 withou...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2012
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368409/ https://www.ncbi.nlm.nih.gov/pubmed/22559222 http://dx.doi.org/10.1021/pr201240a |
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author | Sherrod, Stacy D. Myers, Matthew V. Li, Ming Myers, Jeremy S. Carpenter, Kristin L. MacLean, Brendan MacCoss, Michael J. Liebler, Daniel C. Ham, Amy-Joan L. |
author_facet | Sherrod, Stacy D. Myers, Matthew V. Li, Ming Myers, Jeremy S. Carpenter, Kristin L. MacLean, Brendan MacCoss, Michael J. Liebler, Daniel C. Ham, Amy-Joan L. |
author_sort | Sherrod, Stacy D. |
collection | PubMed |
description | [Image: see text] 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. |
format | Online Article Text |
id | pubmed-3368409 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-33684092012-06-06 Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides Sherrod, Stacy D. Myers, Matthew V. Li, Ming Myers, Jeremy S. Carpenter, Kristin L. MacLean, Brendan MacCoss, Michael J. Liebler, Daniel C. Ham, Amy-Joan L. J Proteome Res [Image: see text] 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. American Chemical Society 2012-05-06 2012-06-01 /pmc/articles/PMC3368409/ /pubmed/22559222 http://dx.doi.org/10.1021/pr201240a Text en Copyright © 2012 American Chemical Society http://pubs.acs.org This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org. |
spellingShingle | Sherrod, Stacy D. Myers, Matthew V. Li, Ming Myers, Jeremy S. Carpenter, Kristin L. MacLean, Brendan MacCoss, Michael J. Liebler, Daniel C. Ham, Amy-Joan L. Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides |
title | Label-Free Quantitation
of Protein Modifications by
Pseudo Selected Reaction Monitoring with Internal Reference Peptides |
title_full | Label-Free Quantitation
of Protein Modifications by
Pseudo Selected Reaction Monitoring with Internal Reference Peptides |
title_fullStr | Label-Free Quantitation
of Protein Modifications by
Pseudo Selected Reaction Monitoring with Internal Reference Peptides |
title_full_unstemmed | Label-Free Quantitation
of Protein Modifications by
Pseudo Selected Reaction Monitoring with Internal Reference Peptides |
title_short | Label-Free Quantitation
of Protein Modifications by
Pseudo Selected Reaction Monitoring with Internal Reference Peptides |
title_sort | label-free quantitation
of protein modifications by
pseudo selected reaction monitoring with internal reference peptides |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368409/ https://www.ncbi.nlm.nih.gov/pubmed/22559222 http://dx.doi.org/10.1021/pr201240a |
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