An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets (MDA-MB-231) during immunological synapse formation.
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ABSTRACT:
SUBMITTER: Perez-Hernandez D
PROVIDER: S-EPMC9944981 | biostudies-literature | 2023 Mar
REPOSITORIES: biostudies-literature
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An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets (MDA-MB-231) during immunological synapse formation.
Perez-Hernandez Daniel D Filali Liza L Thomas Clement C Dittmar Gunnar G
STAR protocols 20230210 1
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 da ...[more]
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Project description:This protocol describes the identification and quantification of phosphopeptides during the dynamic formation of an immunological synapse. Isotope-labeled immune and target cells are mixed, cell-to-cell conjugates are stabilized by cross-linking, and isolated by fluorescence-activated cell sorting (FACS). Phosphopeptides are isolated by phosphopeptide enrichment and measured by mass spectrometry. The resulting data are analyzed to separate cell-specific phosphopeptides using the isotope label and quantified by label-free quantification (LFQ).
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