Gas Phase Separation of Modified Peptides for Activity-Based Protein Profiling

Profiling the reactivity of the proteome with amino-acid level resolution requires the identification and quantification of reacted peptides in the presence of abundant unmodified peptides. Affinity-based approaches use solid sorbents such as streptavidin beads to enrich modified peptides in a solution but are step-intensive and suffer from analyte losses. Here, we describe timShift, a gas phase, in-spectrometer approach for enhanced reactive amino acid profiling, which exploits modification-induced alteration of peptide physical properties. A dicationic, aerodynamic, cysteine-reactive reagent increases the ion mobility of labeled peptides, physically separating them from unmodified 1⁺ and 2⁺ peptide ions and enabling their targeted Sequencing and quantification in whole proteomes. Using these reagents, we profiled >8,200 reactive cysteine sites, demonstrated higher sensitivity versus desthiobiotin/streptavidin enrichment at low protein input, and performed activity-based protein profiling of covalent fragments and selective electrophiles in a 96-well plate format.