Site-specific quantitative cysteine profiling with data-independent acquisition-based mass spectrometry

Chemical proteomics methods, such as activity-based protein profiling, have emerged as powerful and versatile tools to annotate the protein functions and targets of bioactive small molecules in complex biological systems. Incorporated with mass spectrometry (MS)-based quantitative proteomics method, changes of protein activities could be captured and investigated with site-specific precision. However, the semi-stochastic nature of data-dependent acquisition and high cost of the isotopic-labeled reagents make it challenging for chemical biology research to systematically and reproducibly analyze a large number of samples in multidimensional analysis and high-throughput screening. In this chapter, we describe an efficient quantitative chemical proteomic strategy, termed DIA-ABPP, with good reproducibility and high quantification accuracy. Cysteinome profiling was used as a proof-of-concept example with the detailed protocol to demonstrate the workflow of the DIA-ABPP method, including dose-dependent analysis of cysteines that are sensitive to modification by a reactive metabolite, screening of a cysteine-reactive fragment library, and profiling of circadian cysteinome fluctuation. This quantitative chemoproteomic strategy would provide an opportunity for in-depth multi-dimensional chemical proteomic profiling and illuminate the function of bioactive small molecules and proteins in complex biological systems.