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

Area of Science:

• Chemical biology
• Proteomics
• Mass spectrometry

Background:

• Activity-based protein profiling (ABPP) and mass spectrometry (MS) are vital for understanding small molecule interactions and protein functions.
• Current methods face challenges in reproducibility, high cost, and scalability for large-scale, multidimensional analyses.
• Limitations include data-dependent acquisition's stochastic nature and expensive isotopic reagents.

Purpose of the Study:

• To introduce DIA-ABPP, an efficient and reproducible quantitative chemical proteomic strategy.
• To demonstrate the method's high quantification accuracy for complex biological systems.
• To provide a detailed protocol for cysteinome profiling as a proof-of-concept.

Main Methods:

• Developed and validated the DIA-ABPP (Data-Independent Acquisition-Activity-Based Protein Profiling) workflow.
• Utilized MS-based quantitative proteomics for site-specific activity capture.
• Applied cysteinome profiling to showcase dose-dependent analysis, fragment library screening, and circadian fluctuation profiling.

Main Results:

• Achieved good reproducibility and high quantification accuracy with the DIA-ABPP method.
• Successfully demonstrated dose-dependent cysteine modification sensitivity to reactive metabolites.
• Showcased the screening of a cysteine-reactive fragment library and profiling of circadian cysteinome fluctuations.

Conclusions:

• DIA-ABPP offers an efficient strategy for quantitative chemical proteomics.
• The method enables in-depth, multi-dimensional proteomic profiling.
• This approach can illuminate the functions of bioactive small molecules and proteins.