MAPPING ITAM PHOSPHORYLATION SITES BY MASS SPECTROMETRY

通过质谱绘制 ITAM 磷酸化位点

• 批准号: 6137085
• 负责人: KAREN R JONSCHER
• 金额: $ 2.46万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6137085
• 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，某些免疫球蛋白FC的受体 区域和一些病毒蛋白包含一个或多个副本 大约26个氨基酸基矩阵基于免疫受体的酪氨酸 用于与细胞质通信的活化基序（ITAM） 信号转导期间的效应子。 我们建议使用矩阵辅助 激光解吸电离（MALDI）飞行时间（TOF）质量 光谱法直接映射位点并确定化学计量学 含ITAM的传感器成分的磷酸化 B细胞受体复合物，Ig-Alpha和Ig-Beta。 研究将扩展 初步发现IG-Alpha和Ig-Beta Itams中的酪氨酸 在体外由各种SRC家庭激酶不对称地磷酸化 并提示不对称的发现导致独特的生物学 B细胞的反应。 研究将评估不对称的 配体诱导的酪氨酸磷酸化随着B细胞的函数而发生 成熟度和/或抗原亲和力和价值。 不成熟和成熟b 来自转基因3-83只小鼠的细胞将用抗原刺激，裂解 和ITAMS使用抗al-α进行免疫沉淀。 遵循SDS-PAGE 分离和电印迹到硝酸纤维素，代表的条 将切除Ig-Alpha和Ig-beta带，原位酶促消化 将进行，并提取肽。 Maldi-tof Win用于映射 磷酸化位点通过pospossource衰减（PSD）分析。 蛋白质 纯化和质谱参数将使用 合成ITAM肽和嵌合突变体。