Oral Presentation 11th Annual Conference of the International Chemical Biology Society 2022

Development of single-molecule enzyme activity-based protein profiling (SEAP) strategy for liquid biopsy based on ultra-sensitive protein functional analysis (#48)

Toru Komatsu 1 , Shingo Sakamoto 2 , Yu Kagami 2 , Tadahaya Mizuno 1 , Rikiya Watanabe 3 , Kazufumi Honda 4 , Yasuteru Urano 1 5
  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
  2. Cosomil, Inc., Tokyo, Japan
  3. Riken, Saitama, Japan
  4. Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
  5. Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

Cellular functions are mediated by the activities of diverse enzymes, and determining the functional changes during pathogenesis is crucial for understanding and detecting diseases. We have developed various technologies to monitor the changes of enzymatic activities in various pathological states (JACS 2017, Cell Rep. 2021, Chem. Sci. 2022). However, the detection sensitivity of conventional assays for discovering and using enzyme biomarkers in blood samples needs to be improved. In case of DNA and RNA analysis, enhancing the sensitivity of detection to the single-molecule level has revolutionized biomarker discovery and usage. However, the detection methods for proteins, which are thought to contain more functionality-oriented information that can be directly linked to the phenotypes, are yet to attain such a high degree of sensitivity.

In this study, we developed a novel assay platform for comprehensively detecting various enzymes in biological samples at single-molecule level for ultrasensitive and quantitative profiling of the disease-related enzymatic activities. This method is based on single-molecule enzyme analysis performed in a microfabricated chamber device, in which single-molecule enzymes are separately loaded into individual chambers for measuring and detecting thier activities. We developed the system to simultaneously detect various enzyme species in microdevice by establishing a multi substrate-based activity profiling by which each single-molecule enzyme in the chamber is identified in terms of its activity parameters toward multiple fluorogenic substrates. The development of multiple, differentially colored, and reactivity-wide substrates enabled this assay system to discriminately “count” the enzymes in a comprehensive manner (Sci. Adv. 2020).

The single enzyme activity–based protein profiling (SEAP) were applied to detect the activities of alkaline phosphatases (ALPs), ectonucleotide pyrophosphatases/phosphodiesterases (ENPPs), and various peptidases in blood samples at the single-molecule level. As a result, we discovered activities of enzymes that showed significant elevation in blood samples of pancreatic tumor patients, which demonstrates the potential usefulness of this method for finding and using enzyme biomarkers for a highly sensitive and informative liquid biopsy platform.

  1. (1) J. Onagi, T. Komatsu*, et al., J. Am. Chem. Soc. 2017, 139, 3465–3472. (2) K. Yanagi, T. Komatsu*, et al., Cell Rep. 2021, 36, 109311. (3) Y. Kuriki, T. Yoshioka et al., Chem. Sci. 2022, 13, 4474-4481. (4) S. Sakamoto, T. Komatsu*, et al., Science Adv. 2020, 11, eaay0888.