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SP3-FAIMS-Enabled High-Throughput Quantitative Profiling of the Cysteinome
Heta S Desai1,2, Tianyang Yan1,3, Keriann M Backus1,2,3
1Biological Chemistry Department, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
Current Protocols
|July 27, 2022
Summary
This study optimizes chemoproteomic sample preparation using single-pot, solid-phase-enhanced sample preparation (SP3) to improve cysteine analysis. The enhanced workflow increases peptide recovery and enables high-throughput quantitative analysis of the cysteinome.
Area of Science:
- Proteomics
- Chemical Biology
- Analytical Chemistry
Background:
- Chemoproteomic profiling identifies redox-sensitive and ligandable cysteine residues, crucial for functional biology and drug discovery.
- Existing methods face limitations including cumbersome sample preparation, high input material requirements, and low labeled peptide yields.
Purpose of the Study:
- To develop an optimized chemoproteomic sample preparation workflow.
- To enhance peptide labeling and recovery, particularly from small sample volumes.
- To enable high-throughput quantitative analysis of the cysteinome.
Main Methods:
- Integration of enhanced peptide labeling with single-pot, solid-phase-enhanced sample preparation (SP3).
- Customization of the SP3 method for redox proteome analysis.
- Implementation of a tailored workflow within the FragPipe computational pipeline for MS1-based quantification.
Main Results:
- Improved recovery of biotinylated peptides, even from limited sample amounts.
- Successful application of the SP3 platform in multistep sample preparation.
- Accurate MS1-based quantification of peptides, including those with multiple cysteine residues.
Conclusions:
- The optimized workflow significantly enhances high-throughput quantitative analysis of the cysteinome.
- The SP3 platform demonstrates versatility for various chemoproteomic applications.
- This approach facilitates functional biology and drug discovery by improving cysteinome analysis.
