N,N-Dimethylaminopyrene as a fluorescent affinity mass tag for ligand-binding mode analysis

Atsushi Arai,Kozo Yoneda,Yaping Hu,Keita Iio,Hideo Kigoshi,Masaki Kita,Rei Watanabe,Atsunori Hattori

doi:10.1038/s41598-020-64321-9

Atsushi Arai, Kozo Yoneda + Show 6 more

Open Access

https://doi.org/10.1038/s41598-020-64321-9

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Journal: Scientific Reports	Publication Date: Apr 30, 2020
Citations: 4	License type: open-access

Affiliation: Nagoya University, University of Tsukuba

Abstract

Elucidation of the binding mode of protein–ligand interactions provides insights for the design of new pharmacological tools and drug leads. Specific labeling of target proteins with chemical probes, in which the ligands are conjugated with reacting and detecting groups, can establish the binding positions of ligands. Label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) is a promising detection method to selectively detect labeled molecules. However, previous LDI MS tags, such as nitrogen-substituted pyrenes, had problems with low sensitivity and stability. Here we show 6-N,N-dimethylaminopyrene (dmpy) as a versatile mass tag, which was detected at an amount of 0.1 fmol by LA-LDI MS and applicable for MS/MS analysis. By using ligand-dissociation-type dmpy probes and affinity purification with a polystyrene gel, we demonstrated that dmpy-labeled peptides were predominantly detected by MALDI MS. Our dmpy-probe-labeling method might be highly useful for determining the target biomacromolecules of various ligands and their binding sites.

Highlights

Elucidation of the binding mode of protein–ligand interactions provides insights for the design of new pharmacological tools and drug leads
To readily determine the binding mode of target protein–ligand interactions, dmpy, a versatile fluorescent affinity mass tag, has been developed. This tag was detected at an amount of 0.1 fmol by label-assisted laser desorption/ionization (LA-LDI) mass spectrometry (MS), and was useful for peptide MS/MS analysis via effective charge-remote fragmentation
We have aimed at solely detecting pyrene-labeled peptide(s) from a mixture of tryptic peptides by LA-LDI MS22,23

Summary

Introduction

Elucidation of the binding mode of protein–ligand interactions provides insights for the design of new pharmacological tools and drug leads. Many types of mass spectrometry (MS) analyses for biomacromolecules are currently available, such as electrospray ionization (ESI)[7] and matrix-assisted laser desorption/ionization (MALDI)[8,9,10]. These methods are highly sensitive, and PMF is applicable for μg~ng amount of proteins. 42 mu-reduced fragment ions that had lost ketene (CH2=C=O) were observed in association with the parent ions of apy tags To inhibit such in situ fragmentations or oxidative dimerization on the pyrene tag and to further improve the sensitivity of detection for labeled peptides, we have newly developed. We describe LDI MS analysis of dmpy-tag and its application to the precise analysis of target molecule–ligand interactions

Methods

Results

Conclusion