5020
T. H. Truong et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5015–5020
successfully enriched, eluted, and that the biotin moiety can be
separated by TFA treatment. The flexibility of our method provides
options for other model systems that may be more ideally suited
for one type of enrichment method over the other.
Association Scientist Development Award (0835419N to K.S.C.) for
support of this work. The authors also gratefully acknowledge
helpful discussions with Dr. Michael J. Chalmers.
Finally, we applied the strategy outlined in Figure 5(a) to
quantify the relative amounts of oxidized Gpx3 in two different
samples. In the first sample, Gpx3 was oxidized with 0.1 equiv
H2O2 and labeled with d0-DAz-2 or treated with 1.5 equiv H2O2
and labeled with d6-DAz-2. In subsequent steps, heavy and light
DAz-2 probes were removed with gel filtration spin columns,
and the protein samples were combined. Tagged Gpx3 was li-
gated to Yn-ACL, enriched by avidin affinity cartridge or strepta-
vidin-coated magnetic beads, and peptides were analyzed by
LC–MS/MS. Based on our experimental design, we anticipated
that the ratio and intensity of d6-DAz-2-labeled peptide would
be higher relative to the d0-DAz-2-treated sample. The resulting
data from each enrichment method, represented as extracted
ion chromatograms (Fig. 5b and 5d) and single MS scans
(Fig. 5c and 5e), are consistent with this expectation and indicate
that the ratio of d0-DAz-2:d6-DAz-2-tagged peptide is 1:2. The
molecular ion peak at m/z = 765 and 768 correspond to peptides
labeled with d0 or d6-DAz-2 and conjugated to Yn-ACL, with the
loss of one water molecule [M+2HÀH2O]+2. The observed differ-
ence of 3 Da between these ions represents the expected mass
change for the +2 charge state and further MS2 analysis was car-
ried out to confirm the identity of these substituents (Fig. S5a and
S5b). The inset of Figure 5(e) shows the expected decrease in the
heavy-light ratio as the concentration of H2O2 was increased in
d0-DAz-2-treated samples.
To summarize, we have presented a new approach that allows
relative quantification of protein sulfenic acid modifications. This
method utilizes a set of light and heavy sulfenic acid-specific
probes and a complementary acid-cleavable linker to label and en-
rich tagged peptides. Using the prototype thiol peroxidase, Gpx3,
we demonstrate that the biotinylation reagent, Yn-ACL is orthogo-
nal to DAz-2. Biotin-tagged peptides can be enriched via avidin
affinity cartridge or streptavidin-coated magnetic beads, cleaved
with TFA to remove the biotin moiety, and subjected to LC–MS/
MS analysis. A key feature of this approach is that it facilitates
mapping the specific site of cysteine oxidation. Additionally, we
show that the isotope probe pair, d0-DAz-2 and d6-DAz-2, can be
used to monitor relative changes in protein oxidation. Together,
these studies set the stage for global profiling of sulfenic acid for-
mation in H2O2-mediated signaling pathways and disease states
associated with oxidative stress.
Supplementary data
Supplementary data (synthetic procedures, supplementary
data, and protocols for biochemical studies) associated with this
article can be found, in the online version, at doi:10.1016/
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The authors acknowledge funding from the Camile Henry
Dreyfus Teacher Scholar Award (to K.S.C.) and the American Heart