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.