General setup of G-REX: Cells ectopically expressing HaloTag are treated
with the photocaged-RES (i.e., T-REX probe) [in this case, Ht-PreHNE (i.e.,
HaloTag-targetable precursor to HNE)]. HaloTag specifically binds the hexyl
chloride linker of the probe with rapid second-order kinetics[46]. Unbound probe is washed out. Upon photouncaging,
HNE(alkyne), in sub-stoichiometric amount to Halo concentration, is liberated
rapidly inside cells (t1/2 < 1–2
min[32]) within the microenvironment
of Halo, enabling low-occupancy covalent labeling of native KPSs by HNE. Cell
lysis and Click coupling with biotin-azide allows enrichment of HNEylated KPS(s)
by streptavidin pull-down and target-ID enabled by digest LC-MS/MS. Top enriched
targets can be validated using T-REX and they must pass the same level of
stringent negative-control tests (see Fig.
2b legend). Inset: The lifespan of HNE liberated in
G-REX conditions is modeled (Kinetiscope, version 1.1.743.x64).
krelease is the first-order rate constant of
photouncaging. Assuming overall HNE deactivation/degradation follows a
first-order kinetics, kdeact is the corresponding
rate constant.