Antibody-Directed Cell Internalization of Targeted Covalent Europium Tag Enables In Situ Kinase Labeling and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Quantification
Ziyang Fang, Xinyue Zhao, Ruxue Hou, Yang Zhao, Lei Wang, Yi Li, Limin Yang, Xiaowen Yan, Qiuquan Wang
Abstract
Development of methods to accurately detect the expression and activity of protein targets inside live cells is of great significance for disease diagnostics and drug development. Inductively coupled plasma mass spectrometry (ICP-MS) has been demonstrated as a promising technology for protein quantification, the key of which is developing element tags to selectively label the specific protein targets in complex samples with elements sensitive in ICP-MS. Numerous small-molecule element tags (SM-ETs) using DOTA as a lanthanide (Ln) chelating agent have been developed for specific protein target labeling. However, the existing DOTA-based SM-ETs still face limitations including the lack of cell targeting property and low cellular uptake as a result of the inability to penetrate the cell membrane for direct intracellular protein labeling, which restricts the application of SM-ETs for in situ protein quantification. Herein, we present a novel antibody-directed cell/protein dual-targeting strategy that enables us to specifically label and quantify protein targets inside live cells. Based on this strategy, we constructed a multifunctional antibody-targeted covalent Ln tag ( Ab-TC-Ln, LTX-VC-Ibt-DOTA-Eu ), comprising a cell-permeable monoclonal antibody (Loncastuximab, LTX), a targeted covalent Ln tag (Ibt-DOTA-Eu) for Bruton’s tyrosine kinase (BTK), and between them a cathepsin B-cleavable linker (VC). LTX-TC-Ln enables cancer cell recognition, internalization, release of targeted covalent element tags, and specific labeling of Ln to intracellular BTK for ICP-MS quantification. Our work establishes a promising antibody-directed cell internalization strategy for intracellular protein Ln labeling and quantification, which will inspire the development of new ICP-MS approaches toward the quantification of other important protein targets in the future.