Covalent
modification of endogenous proteins by chemical probes
is used for proteome-wide profiling of cellular protein function and
drug discovery. However, probe selectivity in the complex cellular
environment is a challenge, and new probes with better target selectivity
are continuously needed. On the basis of the success of monocovalent
activity-based and reactivity-based probes, an approach of structurally
aligned dual-modifier labeling (SADL) was investigated here on its
potential in improving target precision. Two reactive groups, based
on the acrylamide and NHS ester chemistry, were linked with structural
alignment to be under the same anilinoquinazoline ligand-directive
for targeting the epidermal growth factor receptor (EGFR) protein
kinase as the model system for proteome-wide profiling. The SADL approach
was compared with its monocovalent precursors in a label-free MaxLFQ
workflow using MDA-MB-468 triple negative breast cancer cells. The
dual-modifier probe consistently showed labeling of EGFR with improved
precision over both monocovalent precursors under various controls.
The workflow also labeled endogenous USP34 and PKMYT1 with high selectivity.
Precision labeling with two covalent modifiers under a common ligand
directive may broaden protein identification opportunities in the
native environment to complement genetic and antibody-based approaches
for elucidating biological or disease mechanisms, as well as accelerating
drug target discovery.