MAPPING ITAM PHOSPHORYLATION SITES BY MASS SPECTROMETRY

B和T细胞抗原受体，免疫球蛋白Fc的某些受体 区域和某些病毒蛋白包含一个或多个拷贝的 约26个氨基酸，称为免疫受体酪氨酸 激活基序(ITAM)与细胞质的通讯 信号转导过程中的效应器。我们建议使用矩阵辅助 激光解吸电离飞行时间(TOF)质量 光谱直接绘制位置图并确定化学计量比 含ITAM的转导元件的磷酸化 B细胞受体复合体、Ig-α和Ig-β。这些研究将延长 初步发现Ig-α和Ig-β中的酪氨酸 在体外被不同的Src家族激酶不对称磷酸化 研究结果表明，这种不对称性导致了独特的生物学 B细胞的反应。这些研究将评估不对称 配体诱导的酪氨酸磷酸化是B细胞的一种功能 成熟度和/或抗原亲和力和效价。不成熟和成熟的B 转基因3-83小鼠的细胞将被抗原刺激，裂解 用抗Ig-α免疫沉淀的ITAM。以下是十二烷基硫酸酯-PAGE 硝化棉的分离和电印迹，条带代表 Ig-α和Ig-β条带将被切除，原位酶切 将被执行，并提取多肽。MALDI-TOF WIN用于映射 后震源衰变(PSD)分析的磷酸化位点。蛋白 纯化和质谱学参数将优化使用 合成的ITAM多肽和嵌合突变体。

B and T cell antigen receptors, certain receptors for immunoglobulin Fc regions and some viral proteins contain one or more copies of a approximately 26 amino acid motiftermed immunoreceptor-based tyrosine activation motif (ITAM) utilized for communication with cytoplasmic effectors during signal transduction. We propose to use matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry to directly map the sites and determine the stoichiometry of phosphorylation of the ITAM-containing transducer components of the B cell receptor complex, Ig-alpha and Ig-beta. The studies will extend preliminary findings that the tyrosines in Ig-alpha and Ig-beta ITAMS are asymmetrically phosphorylated by various Src-family kinases in vitro and findings that suggest the asymmetry results in unique biological responses of B cells. The studies will assess whether asymmetrical ligand-induced tyrosine phosphorylation occurs as a function of B cell maturity and/or antigen affinity and valency. Immature and mature B cells from transgenic 3-83 mice will be stimulated with antigen, lysed and ITAMs immunoprecipitated using anti-Ig-alpha. Following SDS-PAGE separation and electroblotting onto nitrocellulose, strips representing Ig-alpha and Ig-beta bands will be excised, in situ enzymatic digests will be performed, and peptides extracted. MALDI-TOF win be used to map phosphorylation sites by postsource decay (PSD) analysis. Protein purification and mass spectrometric parameters will be optimized using synthetic ITAM peptides and chimeric mutants.