Identification of the tyrosine phosphorylation (pY)-dependent interactome of immune co-receptors is crucial for understanding signal pathways involved in immunotherapy. However, identifying the motif-specific interactome for each pY commonly found on these multi-phosphorylated membrane proteins remains challenging. Here, we describe a photoaffinity-based chemical proteomic approach to dissect the motif-specific cytoplasmic interactomes of the critical immune co-receptor CD28. Various full-length CD28 cytoplasmic tails (CD28^cyto) with defined pY and selectively replaced photo-methionine were synthesized and applied to explore three pY-motif-dependent CD28^cyto interactomes. We identified a stand-alone interaction of phospholipase PLCG1 with the Y191 motif with enhanced affinity for the sequence neighboring the transmembrane domain. Importantly, taking advantage of native top-down mass spectrometry with a 193-nm laser, we discovered the direct association of a previously undefined pY218 motif with the kinase PKCθ through its C2 domain. This synthetic CD28^cyto-based photoaffinity proteomic approach is generically applicable to the study of other immune co-receptors with multiple pY sites on their linear cytoplasmic tail.

识别免疫共受体的酪氨酸磷酸化 (pY) 依赖性相互作用组对于了解免疫治疗中涉及的信号通路至关重要。然而，识别这些多磷酸化膜蛋白上常见的每个 pY 的基序特异性相互作用组仍然具有挑战性。在这里，我们描述了一种基于光亲和的化学蛋白质组学方法来剖析关键免疫共受体 CD28 的基序特异性细胞质相互作用。合成了各种具有确定 pY 和选择性替换光甲硫氨酸的全长 CD28 细胞质尾 (CD28^细胞) 并应用于探索三个 pY 基序依赖性 CD28^细胞相互作用组。我们确定了磷脂酶 PLCG1 与 Y191 基序的独立相互作用，对跨膜结构域附近的序列具有增强的亲和力。重要的是，利用带有 193 nm 激光的自上而下质谱法，我们发现了以前未定义的 pY218 基序与激酶 PKCθ 通过其 C2 结构域的直接关联。这种合成的基于 CD28^细胞的光亲和蛋白质组学方法通常适用于在其线性细胞质尾部具有多个 pY 位点的其他免疫共受体的研究。