Ultrasensitive and large-scale protein analysis using single molecule fluorescence gel electrophoresis

2022 Fiscal Year Final Research Report

• Project/Area Number: 19K15718
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 37030:Chemical biology-related
• Research Institution: Kyoto University (2021-2022); Institute of Physical and Chemical Research (2019-2020)
• Principal Investigator: KIM Sooyeon 京都大学, 高等研究院, 客員研究員 (50758886)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Keywords: タンパク質分析 / 電気泳動 / 単一分子蛍光顕微鏡 / 一分子計測 / 一細胞解析

Outline of Final Research Achievements

In this study, we established single-molecule fluorescence gel electrophoresis (single-molecule PAGE) that can count various types of proteins separated by 1D electrophoresis with single-molecule sensitivity using a home-built single-molecule fluorescence microscope. Through the performance evaluation so far, we have confirmed that the detection sensitivity is improved by more than 3 orders of magnitude compared to the conventional staining method (sub-femtogram, attomolar level). In addition, based on this method, we developed single-cell fluorescence gel electrophoresis (single-cell PAGE) for single-cell proteomics analysis. As a result, at least 30 or more protein bands were detected from most of the electropherograms. Furthermore, by using the obtained band patterns, we succeeded in distinguishing different cell lines through dimensionality reduction analysis.

Free Research Field

一分子化学、ケミカルバイオロジ―

Academic Significance and Societal Importance of the Research Achievements

本研究で開発した「一分子蛍光ゲル電気泳動法」は、従来の染色法（CBB、銀染色など）では検出不可であったサブフェムトグラム程度の微量タンパク質を検出でき、現存するタンパク質分析法の中、最も高い感度を持っているといえる。また、「一細胞蛍光電気泳動法」は、一細胞プロテオームを調べる従来法である質量分析や抗体染色より安価かつ簡便であり、さらに、バイアスなく試料内の全タンパク質を調べられるといった利点を持っている。今後、質量分析の前段階や、臨床検査などで一細胞プロテオームを手軽に調べたいという場合に良い選択肢になる可能性が高い。