Evaluation of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Cysteinome Reactivity

Numerous
reagents have been developed to enable chemical proteomic
analysis of small molecule-protein interactomes. However, the performance
of these reagents has not been systematically evaluated and compared.
Herein, we report our efforts to conduct a parallel assessment of
two widely used chemically cleavable linkers equipped with dialkoxydiphenylsilane
(DADPS linker) and azobenzene (AZO linker) moieties. Profiling a cellular
cysteinome using the iodoacetamide alkyne probe demonstrated a significant
discrepancy between the experimental results obtained through the
application of each of the reagents. To better understand the source
of observed discrepancy, we evaluated the key sample preparation steps.
We also performed a mass tolerant database search strategy using MSFragger
software. This resulted in identifying a previously unreported artifactual
modification on the residual mass of the azobenzene linker. Furthermore,
we conducted a comparative analysis of enrichment modes using both
cleavable linkers. This effort determined that enrichment of proteolytic
digests yielded a far greater number of identified cysteine residues
than the enrichment conducted prior to protein digest. Inspired by
recent studies where multiplexed quantitative labeling strategies
were applied to cleavable biotin linkers, we combined this further
optimized protocol using the DADPS cleavable linker with tandem mass
tag (TMT) labeling to profile the FDA-approved covalent EGFR kinase
inhibitor dacomitinib against the cysteinome of an epidermoid cancer
cell line. Our analysis resulted in the detection and quantification
of over 10,000 unique cysteine residues, a nearly 3-fold increase
over previous studies that used cleavable biotin linkers for enrichment.
Critically, cysteine residues corresponding to proteins directly as
well as indirectly modulated by dacomitinib treatment were identified.
Overall, our study suggests that the dialkoxydiphenylsilane linker
could be broadly applied wherever chemically cleavable linkers are
required for chemical proteomic characterization of cellular proteomes.