U.S. Department of Energy

Pacific Northwest National Laboratory

Targeted quantification of protein phosphorylation in the epidermal growth factor receptor signaling pathway

Introduction 

Reversible protein phosphorylation plays an important role in signal transduction.  Dysregulation of protein kinases and phosphatases frequently observed in various diseases further suggests the importanceof accurately characterizing phosphorylation events. MS-based targeted quantification is a promising technology to accurately measure phosphorylation events for specific proteins or pathways. Direct targeted quantification of phosphopeptides in biological samples often suffers from insufficient sensitivity due to the low levels of phosphorylation and high sample complexity. The couple of affinity phosphopeptide enrichment with LC-MS is promising for targeted quantification, but its performance has not been well assessed. Herein, we report an initial assessment of affinity enrichment-targeted quantification workflow and its application for monitoring the dynamic phosphorylation in EGFR pathway.  

Methods

Liquid chromatography-selected reaction monitoring (LC-SRM) was applied to measure phosphopeptide levels quantitatively.  To evaluate the performance of affinity enrichment coupled with LC-SRM for targeted quantification of phosphorylated peptides, we compared immobilized metal ion affinity chromatography (IMAC) and TiO2 enrichment methods in terms of sensitivity, recovery, and reproducibility as measured by LC-SRM.  To illustrate the utility of the approach, this workflow is applied to measure the dynamic phosphorylation changes of 13 key EGFR pathway proteins following stimulation with EGF in a time course experiment. The Waters nanoACQUITY UPLC system and the Thermo Scientific TSQ Vantage were used for LC-SRM measurements. 

Preliminary data 

Although IMAC and TiO2 methods have been widely applied in global discovery phosphoproteomic studies, their performance has not been evaluated for targeted quantification of phosphorylation. We applied LC-SRM to evaluate the sensitivity, recovery, and reproducibility of IMAC- and TiO2-LC-SRM workflow for 34 phosphopeptides in a MCF7 cell digest matrix.  The observed overall recovery ranged from 21-100%, with coefficients of variation of 10-30% depending on the phosphopeptide. IMAC enrichment recovery was more consistent (47-78%) than that of TiO2 enrichment (21-100%) across different phosphopeptides. TiO2 enrichment recovery was more reproducible, with a correlation coefficient of 0.93 vs. 0.68 for IMAC enrichment for different concentrations of phosphopeptides. The sensitivity and reproducibility assessments for these workflows are still ongoing. 

Following these assessment, we applied the optimized workflow to measure the dynamic changes of phosphorylation in EGFR pathway from MCF10A cancer cells after stimulation with 3.0 ng/mL EGF at 0, 10-min, 30-min and 2-hour. A panel of isotopically labeled phosphopeptides for 13 proteins were spiked in 100 µg of tryptic digest from cells at a final concentration of 100 fmol/µL before enrichment as internal standards. Preliminary data demonstrated the quantification of endogenous phosphopeptides for 7 proteins from the sample. The accurate time-dependent phosphorylation levels from cells stimulated with EGF will provide important quantitative mechanistic information for mathematical modeling of this important pathway in cancer research. 

Novel aspect 

A detailed assessment of IMAC- and TiO2-LC-SRM workflows for targeted quantification of protein phosphorylation

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