One-source peptide/phosphopeptide standards for accurate phosphorylation degree determination
Abstract
Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site-specific protein phosphorylation degree determination by nanoLC-ESI-MS/MS. A stable isotope-labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one-source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one-source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.
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The site-specific degree of Tyr-705 phosphorylation of STAT3 at maximal activation is 54.4% at 18 min with 40 ng/mL hIL-6.
“Using quantitative mass spectrometry (Hahn et al., 2011), we determined the site-specific degree of Tyr-705 phosphorylation of STAT3 at the time point of maximal activation (54.4% at 18 min, 40 ng/mL hIL-6; Figure 3C).”
From Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib (2017) · cites this paper as Hahn et al., 2011
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Mass spectrometry–based proteomics turns quantitative
Ong, S.-E., & Mann, M. (2005) · Nature Chemical Biology · doi:10.1038/nchembio736 · Cited by 1,572 · see citing works
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Predictive mathematical models of cancer signalling pathways
Bachmann, J., Raue, A., Schilling, M., Becker, V., Timmer, J., & Klingmüller, U. (2012) · Journal of Internal Medicine · doi:10.1111/j.1365-2796.2011.02492.x · Cited by 63 · see citing works
Self-citationSobotta, S., Raue, A., Huang, X., Vanlier, J., Jünger, A., Bohl, S., Albrecht, U., Hahnel, M. J., Wolf, S., Mueller, N. S., et al. (2017) · Frontiers in Physiology · doi:10.3389/fphys.2017.00775 · Cited by 28 · see citing works
“Using quantitative mass spectrometry (Hahn et al., 2011), we determined the sitespecific degree of Tyr-705 phosphorylation of STAT3 at the time point of maximal activation (54.”
BackgroundSelf-citation
- Authors
- Hahn, B., Böhm, M., Raia, V., Zinn, N., Möller, P., Klingmüller, U., & Lehmann, W.
- Affiliations
- German Cancer Research Center (DE)
- Corresponding author
- Lehmann, Wolf‐Dieter
- Venue
- PROTEOMICS
- Publisher
- Wiley
- Published
- 2011-01-13
- Volume
- 11
- Issue
- 3
- Pages
- 490-494
- ISSN
- 1615-9853 (ISSN-L)
- 1615-9861
- OpenAlex
- W1980037861
- Cited by
- 16
- References
- 1
- Citations by year
- 2024: 1 · 2017: 1 · 2016: 2 · 2015: 1 · 2014: 3 · 2013: 2 · 2012: 3
- FWCI
- 1.17
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- PROTEOMICS (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Helmholtz Association
- German Federal Ministry of Education and Research
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- stable isotope labeling, internal standard, site-specific phosphorylation, dephosphorylation, molar ratio, Stat5a/b, STAT6, signal transduction
- MeSH terms
- Chromatography, Liquid, Hodgkin Disease, Humans, Phosphopeptides, Phosphorylation, Reference Standards, Tumor Cells, Cultured, Spectrometry, Mass, Electrospray Ionization, Tumor Suppressor Proteins, Proteomics, STAT5 Transcription Factor, STAT6 Transcription Factor
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