Development of chemical and rapid identification of the target molecules and ligand binding sites with high resolution

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In this project we aim to develop a method for rapid identification of the target molecules and ligand binding sites with high resolution in the recourse of immunoaffinity purification by anti-ligand antibodies, followed by moderen mass spectrometry. We planed to develop the method for identifying the binding site(s) of glutathione conjugate in cMOAT, an ATP-driven drug trasporter. During the passed 3 years, we obtained the results as follows. (1) We raised a antibody against hapten having analogous structure to photolabeling reagent of TOY-SG, a glutathione derivative. The antibody has a high titer to able to recognize hapten at sub-picomole level. (2) cMOAT protein that was photolabeled by TOY-SG was prepared and purified. We established the conditions of Lys-C digestion, which gave the photolabeled fragments of 3-4 kDa, proper size for MS nalysis. The photolabeled fragments were subjected to immunoaffinify purification using the anti-hapten antibody, but the yield was as low as 〜5%, which was not applicable for further MS analysis. (3) We fractionated the Lys-C fragments by HPLC as an alternative method and the fractions containing photolabeled fragments was analyzed by MALDI-TOF MS. We revealed two new photolabeled sites that located near nucleotide binding sites. The sites had not identified by any other methods such as site-directed mutagenesis. (4) Efforts to raise anti-hapten antibodies with much higher titer are still required.