Quantitative Chemoproteomic Profiling with Data-Independent Acquisition-Based Mass Spectrometry

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 semistochastic nature of data-dependent
acquisition 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. Here, we combine the data-independent
acquisition (DIA) MS with ABPP to develop an efficient label-free
quantitative chemical proteomic method, DIA-ABPP, with good reproducibility
and high accuracy for high-throughput quantification. We demonstrated
the power of DIA-ABPP for comprehensive profiling of functional cysteineome
in three distinct applications, including dose-dependent quantification
of cysteines’ sensitivity toward a reactive metabolite, screening
of ligandable cysteines with a covalent fragment library, and profiling
of cysteinome fluctuation in circadian clock cycles. DIA-ABPP will
open new opportunities for in-depth and multidimensional profiling
of functional proteomes and interactions with bioactive small molecules
in complex biological systems.