Ultrasensitive Detection of Enzymatic Activity Using Single Molecule Arrays

Xu Wang,Alana F Ogata,David R Walt

doi:10.1021/jacs.0c06599

Abstract

Enzyme assays are important for many applications including clinical diagnostics, functional proteomics, and drug discovery. Current methods for enzymatic activity measurement often suffer from low analytical sensitivity. We developed an ultrasensitive method for the detection of enzymatic activity using Single Molecule Arrays (eSimoa). The eSimoa assay is accomplished by conjugating substrates to paramagnetic beads and measuring the conversion of substrates to products using single molecule analysis. We demonstrated the eSimoa method for the detection of protein kinases, telomerase, histone H3 methyltransferase SET7/9, and polypeptide N-acetylgalactosaminyltransferase with unprecedented sensitivity. In addition, we tested enzyme inhibition and performed theoretical calculations for the binding of inhibitor to its target enzyme and show the need for an ultrasensitive enzymatic assay to evaluate the potency of tight binding inhibitors. The eSimoa assay was successfully used to determine inhibition constants of both bosutinib and dasatinib. Due to the ultrasensitivity of this method, we also were able to measure the kinase activities at the single cell level. We show that the eSimoa assay is a simple, fast, and highly sensitive approach, which can be easily extended to detect a variety of other enzymes, providing a promising platform for enzyme-related fundamental research and inhibitor screening.

Highlights

Measuring the activity of enzymes, such as protein kinases, proteases, phosphatases, transferases, and telomerase, is important for many applications including drug discovery, clinical diagnostics and prognosis, and physiological function research.[1−4] Various methods have been developed for the measurement of enzymatic activity and inhibitor screening, including mass spectrometry, electrochemistry, capillary electrophoresis, radiometric methods, colorimetric analysis, and fluorescence methods.[5,6] Colorimetric and fluorescence methods are the most commonly used approaches and enable high-throughput analysis of several thousands of samples per day.[7]
We developed an enzymatic activity using Single Molecule Arrays (eSimoa) assay that enables ultrasensitive detection of enzymes
The background value is 0.0305 ± 0.0022, and the signal to background ratio (S/B) is 63.6 for 1 pM Abl. These results indicate that the eSimoa assay is a powerful method with unprecedented sensitivity that could be used to detect the activity of other protein kinases, such as epidermal growth factor receptor (EGFR) and serine/threonine-protein kinase B-Raf (BRAF)

Summary

INTRODUCTION

Measuring the activity of enzymes, such as protein kinases, proteases, phosphatases, transferases, and telomerase, is important for many applications including drug discovery, clinical diagnostics and prognosis, and physiological function research.[1−4] Various methods have been developed for the measurement of enzymatic activity and inhibitor screening, including mass spectrometry, electrochemistry, capillary electrophoresis, radiometric methods, colorimetric analysis, and fluorescence methods.[5,6] Colorimetric and fluorescence methods are the most commonly used approaches and enable high-throughput analysis of several thousands of samples per day.[7]. To overcome limitations in analytical sensitivity, our laboratory has developed an ultrasensitive detection platform using Single Molecule Arrays (Simoa).[11] target analyte molecules are first captured on biofunctionalized paramagnetic beads. A biotinylated detection probe binds to the captured target analyte molecule on the bead, and the formed complex is labeled with a reporter enzyme streptavidin-β-galactosidase (SβG). The Simoa platform has been used for detecting various analytes such as proteins, small molecules, DNA, and mircoRNAs.[12−15] these assays can only be used for the measurements of analyte concentrations, but not enzymatic activity. Due to the high sensitivity, the eSimoa method was applied to evaluate the potency of tight binding inhibitors This method has been used for determination of protein kinase activity in cell lysates

EXPERIMENTAL SECTION

RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES