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Gas Phase Separation of Modified Peptides for Activity-Based Protein Profiling
Peter Bellotti1, Zirong Chen1,2, Charles D Warren1,2
1Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, New York 10065, United States.
Analytical Chemistry
|July 21, 2025
Summary
We developed timShift, a novel gas-phase method for reactive amino acid profiling. This technique enhances sensitivity and enables precise quantification of modified peptides in complex proteomes.
Area of Science:
- Proteomics
- Chemical Biology
- Analytical Chemistry
Background:
- Profiling proteome reactivity requires identifying modified peptides amidst abundant unmodified ones.
- Current affinity-based enrichment methods are laborious and cause analyte loss.
Purpose of the Study:
- To introduce timShift, an in-spectrometer gas-phase approach for enhanced reactive amino acid profiling.
- To overcome limitations of traditional affinity-based enrichment techniques.
Main Methods:
- Exploiting modification-induced changes in peptide physical properties.
- Utilizing a dicationic, aerodynamic, cysteine-reactive reagent to alter ion mobility.
- Physically separating labeled peptides from unlabeled ones for targeted sequencing and quantification.
Main Results:
- Successfully profiled over 8,200 reactive cysteine sites.
- Demonstrated superior sensitivity compared to desthiobiotin/streptavidin enrichment, especially at low protein input.
- Enabled activity-based protein profiling of covalent fragments and electrophiles in a 96-well plate format.
Conclusions:
- timShift offers a highly sensitive and efficient method for reactive amino acid profiling.
- This gas-phase approach significantly advances proteome reactivity analysis.
- The method facilitates high-throughput activity-based protein profiling.
