Isotopically-Labeled Iodoacetamide-Alkyne Probes for Quantitative Cysteine-Reactivity Profiling

Cysteine
residues on proteins serve a variety of catalytic and
regulatory functions due to the high nucleophilicity and redox activity
of the thiol group. Quantitative proteomic platforms for profiling
cysteine reactivity can provide valuable information related to the
post-translational modification state and inhibitor occupancy of functional
cysteine residues within a complex proteome. Cysteine-reactivity profiling
typically monitors changes in the extent of cysteine labeling by cysteine-reactive
chemical probes, such as iodoacetamide (IA)-alkyne. To enable accurate
measurements of cysteine reactivity changes, isotopic labels are introduced
into the two proteomes of interest using either isotopically tagged
proteomes (SILAC) or cleavable linkers (isoTOP-ABPP) that are installed
using copper-catalyzed azide–alkyne cycloaddition (CuAAC).
Here we provide an alternative strategy for isotopic tagging of two
proteomes for cysteine-reactivity profiling by developing IA-light
and IA-heavy, a pair of isotopically labeled iodoacetamide-alkyne
probes. These probes can be utilized for proteome samples that are
not amenable to SILAC labeling and are facile to synthesize, especially
when compared to the isotopically tagged cleavable linkers. We confirm
the quantitative accuracy of IA-light and IA-heavy by assessing cysteine
reactivity in a purified thioredoxin protein, as well as globally
within a complex proteome where IA-light treatment generates mass-spectrometry
identification of 992 cysteine residues. Importantly, these isotopically
tagged probes can also be utilized for quantifying the percentage
of cysteine modification within a single sample. Preliminary data
supports the use of these tags to quantify the stoichiometry of TCEP-susceptible
cysteine oxidation events in cell lysates.