2. 化合物評価
  3. プロテオーム解析による分子プロファイリング

Molecular Profiling by 2DE-based Proteomic Analysis

(担当:長田裕之、室井 誠)
(Members in charge: Hiroyuki Osada and Makoto Muroi)



In our previous research, we have shown that proteomic change occurs according to the target of the compounds. Cells are treated with compounds provided by the person to be supported and proteomic analysis of lysates of the cells is performed by 2D-DIGE. Comparing the proteomic changes with that of standard compounds in our database, the target of the compounds will be estimated. Practically, the lysates of HeLa cells treated with the compound are analyzed by two-dimensional electrophoresis-based proteomic analysis. Then, using quantitative data, the similarity between the compounds and standard compounds in the database are calculated by multivariate analysis. The target of compound is estimated from the similarity. In addition, significantly changed protein spots caused by the compound is applicable for the estimation of the pathway and the target of the compound.

However, in the case of no target in the HeLa cells or insufficient proteomic change, the estimation of the target will be difficult.


HeLa細胞を培養用シャーレ(6 cm径)にまき込み、翌日サンプルを添加、18時間培養後、回収した細胞を可溶化、2D-DIGEのシステムを用いて296スポットの定量を行う。同時に調製した化合物未処理のコントロールから得られた定量値に対する増減の比を計算し評価に用いる。化合物濃度はHeLa細胞を48時間処理したときにHeLa細胞の増殖を80%以上阻害する濃度で行う。

HeLa cells are plated in a culture dish (6 cm diameter) and the compound is added to the cells on the next day. After 18 h incubation, collected cells are solubilized with buffer and the expression of 296 spots are quantified by 2-D DIGE. The control lysate from HeLa cells treated with DMSO is prepared simultaneously. The fold ratio of each expression spot against that of the control is calculated and used for the evaluation.

Evaluation of compounds


In order to compare the increase/decrease of values with the 80 compounds in our database, multivariate analysis is used. The mechanism of action of the compound is estimated or the novelty of the compound is evaluated. Significantly changed spots among the 296 spots are extracted for the evaluation of the effect on specific pathways.

Desired information (if available)


Effective dose and time treated.


  1. 室井 誠, 長田裕之 “大規模プロファイリングを用いた標的同定”, CSJ Current Review 生物活性分子のケミカルバイオロジー 標的同定と作用機構, 71-78 (2015)
  2. Muroi M, Futamura Y, Osada H. “Integrated profiling methods for identifying the targets of bioactive compounds: MorphoBase and ChemProteoBase.” Nat. Prod. Rep. 33:621-5 (2016)
  3. Muroi M, Kazami S, Noda K, Kondo H, Takayama H, Kawatani M, Usui T, Osada H. “Application of proteomic profiling based on 2D-DIGE for classification of compounds according to the mechanism of action.” Chem. Biol. 17:460-70 (2010)
  4. Muroi M, Osada H. ”Proteomic profiling for target identification of biologically active small molecules using 2D DIGE.” Methods Mol. Biol. 1888:127-39 (2019)