@article{75cfcaf36f254f5bb1fb875d2d7c815e,
title = "An integrated workflow for phosphopeptide identification in natural killer cells (NK-92MI) and their targets (MDA-MB-231) during immunological synapse formation",
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.",
keywords = "Cell Biology, Flow Cytometry/Mass Cytometry, Immunology, Mass Spectrometry, Molecular/Chemical Probes, Protein Biochemistry, Proteomics",
author = "Daniel Perez-Hernandez and Liza Filali and Clement Thomas and Gunnar Dittmar",
note = "Funding Information: C.T. and L.F. are supported by the Luxembourg National Research Fund (ACTIVASION, C19/BM/13579644) and La Fondation Cancer (Luxembourg, ACTIMMUNE, FC/2019/02). We acknowledge the National Cytometry Platform (NCP) for assistance with the single-cell sorting. The NCP is supported by funding from Luxembourg's Ministry of Higher Education and Research (MESR). D.P.H. and L.F. performed the experiments and developed the protocols. C.T. and G.D. conceptualized and supervised the project. D.P.H. and L.F. wrote the manuscript draft. D.P.H. L.F. C.T. and G.D. wrote the final version of the manuscript. The authors declare no competing interests. Funding Information: C.T. and L.F. are supported by the Luxembourg National Research Fund (ACTIVASION, C19/BM/13579644 ) and La Fondation Cancer (Luxembourg, ACTIMMUNE, FC/2019/02 ). We acknowledge the National Cytometry Platform (NCP) for assistance with the single-cell sorting. The NCP is supported by funding from Luxembourg{\textquoteright}s Ministry of Higher Education and Research (MESR). Publisher Copyright: {\textcopyright} 2023 The Authors",
year = "2023",
month = mar,
day = "17",
doi = "10.1016/j.xpro.2023.102104",
language = "English",
volume = "4",
journal = "STAR Protocols",
issn = "2666-1667",
publisher = "Cell Press",
number = "1",
}