Single-Molecule Oxidoreductase Activity Analysis for Activity-Based Diagnosis Based on Proteoform Alterations

Mayano Minoda; Junpei Hatakeyama; Norimichi Nagano; Tadahaya Mizuno; Takumi Iwasaka; Sho Shiga; Kazuki Takahashi; Hideto Hiraide; Shingo Sakamoto; Yu Kagami; Ayumi Kashiro; Kazufumi Honda; Yuki Sugiura; Kazuhiko Mishima; Masayo Kaneko Mishima; Hiroyuki Kusuhara; Yasuteru Urano; Toru Komatsu

doi:10.1021/jacs.4c07624

Single-Molecule Oxidoreductase Activity Analysis for Activity-Based Diagnosis Based on Proteoform Alterations

J Am Chem Soc. 2025 Jan 6. doi: 10.1021/jacs.4c07624. Online ahead of print.

Authors

Mayano Minoda¹, Junpei Hatakeyama², Norimichi Nagano¹, Tadahaya Mizuno¹, Takumi Iwasaka¹, Sho Shiga¹, Kazuki Takahashi², Hideto Hiraide², Shingo Sakamoto^{1

2}, Yu Kagami^{1

2}, Ayumi Kashiro^{3

4}, Kazufumi Honda^{3

4}, Yuki Sugiura^{5

6}, Kazuhiko Mishima⁷, Masayo Kaneko Mishima⁷, Hiroyuki Kusuhara¹, Yasuteru Urano^{1

8}, Toru Komatsu¹

Affiliations

¹ Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
² Cosomil, Inc., Entrepreneur Lab., South Clinical Research Bldg., 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8485, Japan.
³ Institute for Advanced Medical Science, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
⁴ Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
⁵ Center for Cancer Immunotherapy and Immunobiology, Kyoto University Graduate School of Medicine, Rakuyu Kaikan, Yoshida Nihonmatsucho, Sakyo-ku, Kyoto 606-8501, Japan.
⁶ Human Biology Microbiome Quantum Research Center (WPI-Bio2Q), Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
⁷ Department of Neuro-Oncology/Neurosurgery, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka-shi, Saitama 350-1298, Japan.
⁸ Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

PMID: 39762186
DOI: 10.1021/jacs.4c07624

Abstract

We developed a single-molecule enzyme activity assay platform for NAD(P)⁺-dependent oxidoreductases, leveraging a new NAD(P)H-responsive fluorogenic probe optimized for microdevice-based fluorometric detection. This platform enabled the detection of enzyme activities in blood and cerebrospinal fluid (CSF), including lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinases. We demonstrate its potential for activity-based diagnosis by detecting altered populations of enzyme activity species in blood and CSF from liver damage in brain tumor patients.