Activity-based protein profiling (ABPP) has emerged as a powerful and versatile tool to enable annotation of protein functions and discovery of targets of bioactive ligands in complex biological systems. It utilizes chemical probes to covalently label functional sites in proteins so that they can be enriched for mass spectrometry (MS)-based quantitative proteomics analysis. However, the semi-stochastic nature of data-dependent acquisition (DDA) and high cost associated with isotopically encoded quantification reagents compromise the power of ABPP in multidimensional analysis and high-throughput screening, when a large number of samples need to be quantified in parallel.
Recently, Prof. Chu Wang group from College of Chemistry and Molecular Engineering at Peking University integrateda compelling mass spectrometry method, data-independent acquisition (DIA) with ABPP to develop an efficient label-free and site-specific chemoproteomic strategy, named “DIA-ABPP”, for in-depth and large-scale quantitative profiling of reactive residues in proteomes. This new strategy allowed more comprehensive coverage of all the precursor peptide ions regardless of their intensities and showed higher quantitation accuracy with better reproducibility and more comprehensive data coverage.
As proof of concept, the power of DIA-ABPP was demonstrated by comprehensive profiling of functional cysteineome in three distinct applications, including dose-dependent quantification of cysteines’ sensitivity toward a reactive metabolite, screening of ligandablecysteines with a covalent fragment library, and profiling of cysteinome fluctuation in circadian clock cycles. These data not only highlighted the expanded analytical capacity and higher reproducibility of DIA-ABPP, but also provided penetrating biological insights to functional cysteines involved in metabolic regulation and homeostasis maintenance.
As ABPP is a modularized platform depending on the probe design and the biological systems, DIA-ABPP has high compatibility and can be customized in physiological and pathological conditions for different applications and it would be a promising one in analyzing the activity of enzymes and the level of PTMs in a large number of clinical samples in cohort studies, which will greatly promote the studies of disease mechanism and the developments of diagnostic methods.
This work was recently published in the Journal of the American Chemical Society (titled “Quantitative Chemoproteomic Profiling with Data-Independent Acquisition-based Mass Spectrometry”, DOI: 10.1021/jacs.1c11053). The first author is Dr. Fan Yang and the corresponding author is Prof.Chu Wang. This research was funded in part by the Ministry of Science and Technology, National Natural Science Foundation of China, Beijing National Laboratory for Molecular Science, Key Laboratory of Bioorganic Chemistry and Molecular Engineering. Prof. Chu Wang is also an affiliated member of Center for Life Sciences at Peking University.
Original link for the paper: https://pubs.acs.org/doi/pdf/10.1021/jacs.1c11053
