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Published on: August 2, 2018
Enrichment of Tyrosine Phosphorylated Peptides for Quantitative Mass Spectrometry Analysis of RTK Signaling Dynamics
Vidyasiri Vemulapalli1,2, Stephen C Blacklow1,2, Steven P Gygi3
1Department of Cancer Biology, Dana-Farber Cancer Institute Boston, MA 02115, USA.
Abstract:
Cells sense and respond to mitogens by activating a cascade of signaling events, primarily mediated by tyrosine phosphorylation (pY). Because of its key roles in cellular homeostasis, deregulation of this signaling is often linked to oncogenesis. To understand the mechanisms underlying these signaling pathway aberrations, it is necessary to quantify tyrosine phosphorylation on a global scale in cancer cell models. However, the majority of the protein phosphorylation events occur on serine (86%) and threonine (12%) residues, whereas only 2% of phosphorylation events occur on tyrosine residues ( Olsen et al., 2006 ). The low stoichiometry of tyrosine phosphorylation renders it difficult to quantify cellular pY events comprehensively with high mass accuracy and reproducibility. Here, we describe a detailed protocol for isolating and quantifying tyrosine phosphorylated peptides from drug-perturbed, growth factor-stimulated cancer cells, using immunoaffinity purification and tandem mass tags (TMT) coupled with mass spectrometry.
Insights
This study presents a new protocol to quantify tyrosine phosphorylation (pY) in cancer cells. The method uses immunoaffinity purification and mass spectrometry to overcome challenges in measuring low-abundance pY events.
Area of Science:
- Molecular Biology
- Cell Signaling
- Proteomics
Background:
- Cellular responses to mitogens involve signaling cascades, primarily mediated by tyrosine phosphorylation (pY).
- Aberrant tyrosine phosphorylation signaling is frequently implicated in oncogenesis and cancer development.
- Quantifying global tyrosine phosphorylation is crucial for understanding cancer signaling but challenging due to its low stoichiometry (2% of all phosphorylation events).
Purpose of the Study:
- To develop and describe a robust protocol for the global quantification of tyrosine phosphorylated peptides in cancer cells.
- To enable comprehensive analysis of tyrosine phosphorylation events, even at low stoichiometry, in response to stimuli like growth factors and drugs.
Main Methods:
- Immunoaffinity purification was employed to enrich tyrosine phosphorylated peptides.
- Tandem Mass Tags (TMT) labeling was utilized for quantitative proteomic analysis.
- Mass spectrometry was coupled with these techniques for high-accuracy identification and quantification.
Main Results:
- A detailed protocol was established for isolating and quantifying tyrosine phosphorylated peptides.
- The method allows for comprehensive analysis of tyrosine phosphorylation events in drug-perturbed, growth factor-stimulated cancer cells.
- The protocol addresses the challenges posed by the low abundance of tyrosine phosphorylation.
Conclusions:
- The described protocol provides a reliable method for global tyrosine phosphorylation quantification in cancer research.
- This technique facilitates a deeper understanding of signaling pathway aberrations linked to cancer.
- The method enhances the ability to study tyrosine phosphorylation dynamics in complex biological systems.
