Department of Infection and Immunity, Luxembourg Institute of Health, Strassen, Luxembourg.
Department of Cancer Research, Luxembourg Institute of Health, Strassen, Luxembourg.
STAR Protoc. 2023 Mar 17;4(1):102104. doi: 10.1016/j.xpro.2023.102104. Epub 2023 Feb 10.
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.
在这里，我们提出了一个方案，以鉴定和定量在免疫突触的动态形成过程中的磷酸肽。我们描述了混合同位素标记的免疫和靶细胞的步骤，通过交联稳定细胞 - 细胞偶联物，并用荧光激活细胞分选分离它们。我们详细描述了通过磷酸肽富集分离磷酸肽，并通过质谱法对其进行后续测量。最后，我们描述了使用同位素标记和无标记定量法分离细胞特异性磷酸肽的数据分析。
