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Updated: Aug 8, 2025
Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer
Published on: February 11, 2015
An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets
Daniel Perez-Hernandez1, Liza Filali2, Clement Thomas2
1Department of Infection and Immunity, Luxembourg Institute of Health, Strassen, Luxembourg.
Insights
This study introduces a new method to track dynamic phosphopeptide changes during immune cell interactions. The protocol enables precise identification and quantification of phosphopeptides involved in immunological synapse formation.
Area of Science:
- Immunology
- Cell Biology
- Proteomics
Background:
- The immunological synapse is crucial for adaptive immune responses.
- Understanding dynamic signaling events within the synapse is challenging.
- Phosphorylation plays a key role in immune cell communication.
Purpose of the Study:
- To develop a robust protocol for identifying and quantifying phosphopeptides during immunological synapse formation.
- To enable detailed analysis of dynamic phosphorylation events in cell-cell interactions.
Main Methods:
- Utilizing isotope-labeled immune and target cells for differential labeling.
- Stabilizing cell conjugates via chemical cross-linking.
- Isolating specific cell populations using fluorescence-activated cell sorting (FACS).
- Enriching phosphopeptides using affinity-based methods.
- Quantifying phosphopeptides via mass spectrometry (MS).
- Analyzing data using label-based and label-free quantification strategies.
Main Results:
- The protocol successfully identifies and quantifies phosphopeptides in a dynamic cellular context.
- Distinguishes cell-specific phosphopeptide profiles within the synapse.
- Provides a quantitative measure of phosphorylation changes during synapse formation.
Conclusions:
- This protocol offers a powerful tool for dissecting signaling pathways in immune cell interactions.
- It facilitates a deeper understanding of the molecular mechanisms governing the immunological synapse.
- The method is adaptable for studying other dynamic cell-cell communication processes.
Abstract:
Here, we present a protocol to identify and quantify phosphopeptides during the dynamic formation of an immunological synapse. We describe steps for mixing isotope-labeled immune and target cells, the stabilization of cell-to-cell conjugates by cross-linking, and their isolation by fluorescence-activated cell sorting. We detail the isolation of phosphopeptides by phosphopeptide enrichment and their subsequent measurement by mass spectrometry. Finally, we describe the analysis of the resulting data to separate cell-specific phosphopeptides using the isotope label and label-free quantification.
