MAPPING ITAM PHOSPHORYLATION SITES BY MASS SPECTROMETRY

通过质谱绘制 ITAM 磷酸化位点

• 批准号: 2520844
• 负责人: KAREN R JONSCHER
• 金额: $ 2.5万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-13 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2520844
• 关键词: B lymphocyte; T cell receptor; leukocyte activation /transformation; mass spectrometry; method development; phosphoproteins; phosphorylation; protein structure function; stoichiometry

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.

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。