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

开发高分辨率的化学和快速鉴定靶分子和配体结合位点的方法

• 批准号: 12470483
• 负责人: NAKAYAMA Hitoshi
• 金额: $ 8.64万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470483/
• 关键词: photoaffinity labeling; anti-peptide antibodies; TOF-MS; ESI-MS; MS; photolabeled peptide fragments; identifying binding sites; TDF-MS; ラベル化ペプチド解析; 神経特異的ハチ青

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.

在这个项目中，我们旨在开发I方法，以快速识别抗球形抗体的降压位置，并具有高分辨率，然后使用Moderen质谱法。谷胱甘肽在CMOAT中的结合物，ATP-drug trasporter是一种抗体，它具有与Toy-SG的相似结构，该抗体是谷胱甘肽衍生物的抗体。 SG制备并纯化。低至〜5％，这不适用于进一步的MS分析（3）我们分馏了Lys-C碎片方法，并通过MALDI-TOF MS分析了分级的光片片段。结合坐着。

项目成果

A.Kuniyasu, et al.: "CD36-mediated endocytic uptake of AGE in mouse 3T3-L1 and human subcutaneous adipocytes"FEBS Lett.. 537. 85-90 (2003)

A.Kuniyasu 等人：“CD36 介导的小鼠 3T3-L1 和人皮下脂肪细胞中 AGE 的内吞摄取”FEBS Lett.. 537. 85-90 (2003)

K.Kawahara, et al.: "Induction of CHOP and apoptosis by nitric oxide in p53-deficient microglial cells"FEBS Lett.. 506(2). 135-139 (2001)

K.Kawahara 等人：“p53 缺陷型小胶质细胞中一氧化氮诱导 CHOP 和细胞凋亡”FEBS Lett.. 506(2)。

K.Kawahara, et al.: "Efficient identification of photolabeled amino acids by mmuno-purification and mass spectrometry"Biochem. J.. 363(2). 223-232 (2002)

K.Kawahara 等人：“通过免疫纯化和质谱法有效鉴定光标记氨基酸”Biochem。

N.Ohgami et al.: "Glibenclamide acts as an inhibitor of acyl-CoA : cholesterol acyltransferase enzyme"Biochem.Biophys.Res.Commun.. 277. 417-422 (2000)

N.Ohgami 等人：“格列本脲作为酰基辅酶 A 的抑制剂：胆固醇酰基转移酶”Biochem.Biophys.Res.Commun. 277. 417-422 (2000)

A.Kuniyasu, et al.: "Adipocytes recognize and degrade oxidized LDL through CD36"Biochem. Biophys. Res. Commun.. 295(2). 319-323 (2002)

A.Kuniyasu 等人：“脂肪细胞通过 CD36 识别并降解氧化的 LDL”Biochem。