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

Daniel Perez-Hernandez*, Liza Filali, Clement Thomas, Gunnar Dittmar*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

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.

Original languageEnglish
Article number102104
JournalSTAR Protocols
Volume4
Issue number1
Early online date9 Feb 2023
DOIs
Publication statusPublished - 17 Mar 2023

Keywords

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

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