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Title: Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway

Abstract

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cellsmore » following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.« less

Authors:
; ; ; ; ; ; ; ORCiD logo; ORCiD logo; ; ORCiD logo
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1439027
Report Number(s):
PNNL-SA-131519
Journal ID: ISSN 0003-2700; 50077; 453040220
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry; Journal Volume: 90; Journal Issue: 8
Country of Publication:
United States
Language:
English
Subject:
Environmental Molecular Sciences Laboratory

Citation Formats

Yi, Lian, Shi, Tujin, Gritsenko, Marina A., X’avia Chan, Chi-Yuet, Fillmore, Thomas L., Hess, Becky M., Swensen, Adam C., Liu, Tao, Smith, Richard D., Wiley, H. Steven, and Qian, Wei-Jun. Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway. United States: N. p., 2018. Web. doi:10.1021/acs.analchem.8b00071.
Yi, Lian, Shi, Tujin, Gritsenko, Marina A., X’avia Chan, Chi-Yuet, Fillmore, Thomas L., Hess, Becky M., Swensen, Adam C., Liu, Tao, Smith, Richard D., Wiley, H. Steven, & Qian, Wei-Jun. Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway. United States. doi:10.1021/acs.analchem.8b00071.
Yi, Lian, Shi, Tujin, Gritsenko, Marina A., X’avia Chan, Chi-Yuet, Fillmore, Thomas L., Hess, Becky M., Swensen, Adam C., Liu, Tao, Smith, Richard D., Wiley, H. Steven, and Qian, Wei-Jun. Tue . "Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway". United States. doi:10.1021/acs.analchem.8b00071.
@article{osti_1439027,
title = {Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway},
author = {Yi, Lian and Shi, Tujin and Gritsenko, Marina A. and X’avia Chan, Chi-Yuet and Fillmore, Thomas L. and Hess, Becky M. and Swensen, Adam C. and Liu, Tao and Smith, Richard D. and Wiley, H. Steven and Qian, Wei-Jun},
abstractNote = {Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.},
doi = {10.1021/acs.analchem.8b00071},
journal = {Analytical Chemistry},
number = 8,
volume = 90,
place = {United States},
year = {Tue Mar 27 00:00:00 EDT 2018},
month = {Tue Mar 27 00:00:00 EDT 2018}
}