Identification of SH3-domain Binding Proteins from Cell and Tissue Extracts by Using Peptide-Mass Fingerprinting

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Efs is a Fyn-binding protein with an SH3 domain and multiple tyrosine phosphorylation sites,and it functions as a possible modulator in intracellular signal transduction.To better understand signaling through Efs,we searched for Efs-binding proteins by using immobillized SH3 domains fused to glutathione S-transferase (GST).Proteins which bound to the affinity matrices from cell and tissue extracts were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).By this method,it was possible to identify multiple at one time.However,special care was necessary to eliminate nonspecific blinding of cytoskeletal proteins,including actin,myosin and tropomyosin;the amounts of these proteins present in the cell lysate were large and signaficant amounts were found to bind to the affinity matrices. The preclearing process did not work well to eliminate these cyroskeletal fractions and a specific elution procedure was necessary to selectively identify signaling proteins.
札幌医科大学の論文