MAPPING ITAM PHOSPHORYLATION SITES BY MASS SPECTROMETRY

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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.