An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets (MDA-MB-231) during immunological synapse formation

Abstract

Summary: 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Flow Cytometry/Mass Cytometry
• Immunology
• Molecular/Chemical Probes
• Protein Biochemistry
• Proteomics