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

本课题的目的是建立一种通过抗配体抗体的免疫亲和纯化，结合现代质谱技术，以高分辨率快速鉴定靶分子和配体结合位点的方法。我们计划建立一种方法来鉴定ATP驱动的药物转运体cMOAT中谷胱甘肽结合物的结合位点。在过去的三年里，我们取得了以下成果。(1)我们提出了一种抗半抗原的抗体，其结构类似于谷胱甘肽衍生物TOY-SG的光标记试剂。该抗体具有高滴度，能够识别亚皮摩尔水平的半抗原。(2)制备并纯化TOY-SG光标记的cMOAT蛋白。确定了Lys-C酶切条件，得到分子量为3- 4kDa的光标记片段，大小适宜于质谱分析。使用抗半抗原抗体对光标记的片段进行免疫亲和纯化，但产率低至1.5%，这不适用于进一步的MS分析。(3)我们分馏的Lys-C片段通过HPLC作为一种替代方法，并含有光标记的片段的馏分进行了分析，通过MALDI-TOF MS。我们发现了两个新的光标记的网站，位于附近的核苷酸结合位点。这些位点没有通过任何其他方法如定点突变来鉴定。(4)仍然需要努力提高具有高得多的滴度的抗半抗原抗体。

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.