Harnessing Redox Cross-Reactivity to Profile Distinct Cysteine Modifications

Article abstract of DOI:10.1021/jacs.5b06806

Cysteine S-nitrosation and S-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous S-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of S-nitrosation. In parallel, S-nitrosothiol-linked probes enable enrichment and detection of endogenous S-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases S-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both S-nitrosation and S-sulfination. Quantitative proteomic analysis demonstrates that both S-nitrosation and S-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids.

Full text of DOI:10.1021/jacs.5b06806

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Article
Harnessing redox cross-reactivity to profile distinct cysteine modifications
Jaimeen D. Majmudar, Aaron M Konopko, Kristin J. Labby, Christopher
T.M.B. Tom, John E Crellin, Ashesh Prakash, and Brent R. Martin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b06806 • Publication Date (Web): 19 Jan 2016
Downloaded from http://pubs.acs.org on January 19, 2016
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accumulated Sꢀsulfination . Additionally, Sꢀsulfination
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of the PARK7 (DJꢀ1) is enigmatically critical for the
protein’s redox chaperone activity . While there are no
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reported methods for mass spectrometry profiling of Sꢀ
sulfination, recently reported substituted arylꢀnitroso
probes suggest Sꢀsulfination is widespread, and may
play a broader role in protein structure, redox homeostaꢀ
22
sis, and cellular regulation .
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Here we explore the cross reactivity of Sꢀsulfination
with Sꢀnitrosation, which react to form a stable thiosulꢀ
fonate. Biotin conjugated probes enable reciprocal deꢀ
tection, enrichment, and analysis of each redox postꢀ
translational modification in cell and tissue homogeꢀ
nates, and suggests a broader role for sulfinic acids in
redox regulation.
Results and Discussion
Sulfinic acids react selectively with S-nitrosothiols.
While exploring the interplay of cysteine postꢀ
translational modifications, we identified a reported reꢀ
action between phenylsulfinic acid and Sꢀ
nitrosocysteine, leading to thiosulfonate formation in
2
3
physiological
buffers
at
room
temperature .
Phenylsulfinic acid reacts rapidly with NꢀacetylꢀSꢀ
nitrosoꢀcysteine methyl ester to yield a thiosulfonate
product (Figure 1a). More than 99% of the thiosulfonate
product remained after 5 hours at pH ≤ 7, demonstrating
robust stability in physiological buffers (Figure S1).
Moreover, we did not observe any additional products
formed using a photodiode array detector, suggesting a
direct conversion of reactants to products.
Thiosulfonates are readily exchangeable with thiols,
serving as the basis for the cysteine capping agent meꢀ
24
thyl methanethiosulfonate (MMTS) . To prevent such
exchange, we found that sulfinic acids do not react with
iodoacetamide (IAM) (Figure 1b and Figure S2a) or
cysteine (Figure S2b) in aqueous buffers, enabling orꢀ
thogonal alkylation of thiols without perturbing nitroꢀ
sothiols or sulfinic acids. Interestingly, a large number of
reported Sꢀnitrosation studies alkylate thiols with
25
MMTS , which releases methylsulfinic acid upon reacꢀ
tion with cysteine. Any released methylsulfinic acid may
proceed to react with Sꢀnitrosothiols, potentially reducꢀ
26
ing Sꢀnitrosation detection .
Approximately 6ꢀ10% of all cellular thiols are oxidized
and engaged in a disulfide bonds, which can rise to
Figure 1. Sulfinic acid reactivity in phosphate buffer. (a)
Phenylsulfinic acid (2, 20 mM) reacts with Nꢀacetyl Sꢀnitroso
cysteine methyl ester (1, 5 mM) to form thiosulfonate 3.
Absorbance was measured at 283 nm. (b) No additional peaks are
observed when phenylsulfinic acid is incubated with
iodoacetamide in PBS for 30 minutes. Absorbance was measured
at 291 nm. (c) Phenylsulfinic acid, 2, does not react with the
activated disulfide DTNB, 5. Absorbance was measured at 291
nm for phenylsulfinic acid and 265 nm for DTNB and the reaction
mixture.
2
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>15% upon oxidative stress . We find that phenylsulfinꢀ
ic acid does not react with biologically relevant disulꢀ
fides such as cystine (Figure S2c) or activated disulfides
such as 5,5’ꢀdithiobisꢀ(2ꢀnitrobenzoic acid), DTNB
(Figure 1c). However, the highly activated disulfide
dipyridyldisulfide (aldrithiol) forms an insoluble species
when mixed with equimolar phenylsulfinic acid, but
does partially react overnight in a 50% DMSO / PBS
solution (data not shown). Finally, we observe no
reaction between phenylsulfinic acid and benzaldehyde
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(
Figure S2d) or with pyrrolidinone sulfenamide (Figure
Nꢀhydroxysulfonamides are prone to degradation at
S3). Overall, sulfinic acids do not react with free thiols,
biological disulfides, aldehydes or sulfenamides, highꢀ
lighting an unappreciated chemoselective reaction with
Sꢀnitrosothiols. Furthermore, after initial iodoacetamide
alkylation of free thiols, sulfinic acid probes could be
leveraged to label and enrich Sꢀnitrosothiols for analysis.
higher pH, decomposing to release sulfinic acid and niꢀ
31
troxyl (HNO) . To investigate whether the subꢀ
stoichiometric formation of the Nꢀhydroxysulfonamide
product can be attributed to loss through degradation, we
assayed
the
stability
of
Nꢀhydroxyꢀ4ꢀ
methylbenzenesulfonamide at pH 1, 4, 7 and 10 (Figure
S5e). Our findings demonstrate that Nꢀhydroxyꢀ4ꢀ
methylbenzenesulfonamide is stable at pH 1, and deꢀ
grades slowly at pH 4. At pH 7, Nꢀhydroxyꢀ4ꢀ
methylbenzenesulfonamide decomposes with a halfꢀlife
of ~4 hours. Furthermore, since the decomposition of Nꢀ
hydroxyꢀ4ꢀmethylbenzenesulfonamide generates nitroxꢀ
yl and a sulfinic acid, excess sulfinic acid will slow deꢀ
Reaction analysis. To characterize the reaction of niꢀ
trosothiols and sulfinic acids, we assayed thiosulfonate
formation by measuring the loss of Sꢀnitrosoꢀglutathione
(GSNO) absorbance at 340 nm after phenylsulfinic acid
addition (Figure S4). Increasing amounts of sodium
phenylsulfinate were titrated to a 2 mM solution of Sꢀ
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nitrosoglutathione in the dark. The reported pK of a
a
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composition by mass action . After taking degradation
into account, Nꢀhydroxysulfonamide formation is not
stoichiometric with formation of the thiosulfonate prodꢀ
uct. Overall, the reaction between sulfinic acids and niꢀ
trosothiols proceeds by an alternative mechanism withꢀ
out concomitant Nꢀhydroxysulfonamide formation.
sulfinic acid is ~2.8 , yet the reaction proceeds similarly
at pH 1, suggesting the sulfur lone pair acts as the nucleꢀ
ophile, independent of the protonation state. Sulfinic
acids are ambident nucleophiles , where the soft sulfur
atom is the attacking species, and the oxygen charge
state should not significantly affect sulfur nucleophiliciꢀ
ty. At pH 1, 4, and 7, the reaction rate is approximately
first order and proceeds at 3 x 10 M s . At neutral pH,
the concentration dependence is slightly hyperbolic,
suggesting a small contribution from an alternate reacꢀ
tion mechanism, and no reaction occurs under basic
conditions.
29
Biotin-SO H detects S-nitrosated proteins. Next, we
2
ꢀ2
ꢀ1 ꢀ1
examined the reactivity of reporter linked sulfinic acids
with Sꢀnitrosated proteins. Biotin and fluorescein Nꢀ
hydroxysuccinimide (NHS) esters were directly coupled
to the sulfinic acid metabolite hypotaurine (Biotinꢀ
SO H) or the sulfonic acid metabolite taurine (Biotinꢀ
2
SO H) in deꢀgassed water, purified by preparative
3
HPLC, and stored in singleꢀuse aliquots at ꢀ80 °C to
prevent oxidation. BiotinꢀSO H is stable during the duꢀ
2
ration of labeling (< 1 hour), but is fully oxidized to bioꢀ
tinꢀSO H after ~5 hours in atmospheric oxygen.
3
Each probe was incubated with mammalian cell lyꢀ
sates denatured in 6 M urea in phosphateꢀbuffered saline
(PBS), preꢀalkylated with excess iodoacetamide, and
analyzed by nonꢀreducing SDSꢀPAGE and streptavidinꢀ
Cy5 blot detection. BiotinꢀSO H, but not biotinꢀSO H,
2
3
labeled a rich profile of proteins (Figure 3a), confirming
the reaction is dependent on the nucleophilic sulfinic
Figure 2. Reaction kinetics and byꢀproduct analysis. (a) Reaction
rate between phenylsulfinic acid and GSNO at various pHs. (b)
Percent yield of the thiosulfonate product and the 4ꢀmethylꢀ
Piloty’s acid product.
acid. BiotinꢀSO H labeling is enhanced at higher probe
2
concentrations (Figure S6a) or by preꢀincubation with
the nitric oxide donor methylamine hexamethylene meꢀ
thylamine NONOate (MAHMAꢀNONOate) (t1/2 = 3
min) (Figure 3b and S6b). Furthermore, labeling is
eliminated by pretreatment with excess hypotaurine
Figure S6c), demonstrating saturated labeling above 5
mM. In addition, 365 nm ultraviolet photolysis of Sꢀ
nitrosothiols or pretreatment with ascorbate eliminates
A potential reaction mechanism was recently reported
requiring two sulfinic acids; one to attack the nitrogen of
the nitrosothiol, and a second sulfinic acid to react with
the sulfur to displace Nꢀhydroxysulfonamide and the
(
30
thiosulfonate in stoichiometric amounts . This is in conꢀ
trast to our initial reaction analysis, which did not reveal
any additional products. Standard curves were then deꢀ
rived from isolated chemical standards to allow detailed
analysis of the reaction between sodium 4ꢀmethylꢀ
phenyl sulfinate and GSNO (Figure S5a-d). This analyꢀ
sis reveals subꢀstoichiometric formation of Nꢀ
hydroxybenzenesulfonamide, which is only detected in
the presence of significant excess sulfinic acid (Figure
biotinꢀSO H labeling (Figure 3c-d). Importantly, all
2
biotinꢀSO H labeling is all labeling is thiolꢀdependent,
2
and reversed by postꢀincubation with tris(2ꢀ
carboxyethyl)phosphine (TCEP).
Sulfenic acids (RꢀSOH) describe the metaꢀstable oxiꢀ
dation of thiols to a highly reactive intermediate, which
are typically resolved by a secondary thiol during disulꢀ
2
b), and thus not in our initial nearꢀstoichiometric HPLC
18a, 33
fide formation
. Based on the electrophilic nature of
analysis.
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sulfenic acids, we next examined if sulfinic acids react
remove any sulfenic acids, limiting any concerns about
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with sulfenic acids. The sulfenic acid sensitive probe
dimedone is widely used to covalently trap sulfenic acꢀ
ids in complex proteomes and in living systems . In
order to explore this potential crossꢀreactivity, we found
that standard working concentrations of dimedone had
crossꢀreactivity. In addition, ascorbate eliminates nearly
all dimedone labeling, providing further evidence in the
absence of denaturing buffers, sulfenic acids may also
be analyzed when using the biotinꢀswitch method for
detecting Sꢀnitrosation. In addition, sodium metaꢀ
arsenite has been reported as a selective sulfenic acid
3, 34
no effect on biotinꢀSO H labeling in cell lysates, conꢀ
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3
5
firming dimedone does not significantly interfere with Sꢀ
nitrosothiols. Interestingly, dimedoneꢀalkyne labeling
was largely eliminated after denaturing proteins in 6 M
reductant . We do not observe any arseniteꢀdependent
effects on biotinꢀSO H labeling (Figure S7), providing
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further support for orthogonal labeling between sulfinic
acids and nitrosothiols.
urea. Thus, the denaturing biotinꢀSO H assay conditions
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Figure 3. Sulfinic acid probes selectively label Sꢀnitrosothiols in HEK293T cell lysates. Unless otherwise noted, all lysates are denatured
in 6 M urea supplemented with 50 mM iodoacetamide (IAM). (a) BiotinꢀSO H, but not biotinꢀSO H, labels Sꢀnitrosothiols in lysates. (b)
2
3
BiotinꢀSO H labeling increases following preꢀtreatment with MAHMA NONOate, a nitric oxide donor before IAM addition. (c) UV
2
photolysis (365 nm) preꢀtreatment eliminates biotinꢀSO H labeling. (d) BiotinꢀSO H labeling is eliminated by preꢀtreatment with
2
2
ascorbate. (e) The products of biotinꢀSO H labeling are sensitive to postꢀtreatment by the reductant TCEP. (f) The sulfenic acids probe
2
dimedone does not reduce biotinꢀSO H labeling. (g) Denaturing buffers or ascorbate reduce dimedoneꢀalkyne labeling of sulfenic acids.
2
Following a 1 hour incubation with dimedone alkyne, lysates were chloroform/methanol precipitated and mixed with TBTA, CuSO4,
TCEP, and TAMRAꢀazide for 1 hour in PBS before gel analysis. (h) MMTS and IAM both react with free thiols, but MMTS liberates
methane sulfinic acid and interferes biotinꢀSO H labeling of Sꢀnitrosated proteins.
2
The common alkylating agent, methyl methane thioꢀ
sulfonate (MMTS) reacts with free thiols to form a diꢀ
sulfide bond, releasing methane sulfinic acid. Surprisꢀ
ingly, if we block thiols with MMTS instead of iodoaꢀ
for the detection of endogenous Sꢀnitrosothiols.
cetamide, we observe a complete loss in biotinꢀSO H
2
labeling (Figure 3h and Figure S8a). Based on this
analysis, sufficient methyl sulfinic acid is released
through MMTS alkylation of thiols to react with nitroꢀ
Figure 4. Labeling of recombinant human GAPDH with biotinꢀ
2
6
sothiols . This effect could similarly be caused by transꢀ
nitrosation between thiol contaminants formed through
SO H. (a) GAPDH labeling is observed only in the presence of
2
MAHMA NONOate, and eliminated by preꢀtreatment with
ascorbate. (b) GAPDH is labeled by the transꢀnitrosation donor
GSNO, and eliminated by preꢀtreatment with the reducing agent
DTT.
36
slow disproportionation of sulfinic acids . This is unꢀ
likely, since removing MMTS prior to biotinꢀSO H does
2
not restore labeling (Figure S8b). These findings warꢀ
rant a careful reinterpretation of the biotinꢀswitch Sꢀ
nitrosation enrichment method, which typically begins
with MMTS alkylation of free thiols. Once nitrosothiols
react with methyl sulfinic acid, the resulting thiosulꢀ
fonate can be slowly reduced by ascorbate, liberating a
free thiol for further enrichment. Overall, iodoacetamide
effectively blocks all thiols in our experiments, as well
Next, biotinꢀSO H was used to profile the Sꢀ
2
nitrosation of purified recombinant human glyceraldeꢀ
hyde 3ꢀphosphate dehydrogenase (GAPDH), a known Sꢀ
3
8
nitrosated protein . GAPDH was dissolved in PBS and
then treated under different conditions, followed by inꢀ
cubation with iodoacetamide and biotinꢀSO H to primarꢀ
2
ily evaluate the specificity of sulfinic acid probes for
nitrosothiols using a purified protein. As expected, no Sꢀ
37
as sites of persulfidation , providing a robust platform
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nitrosation was observed until treatment with MAHMA
NONOate, which was reduced by ascorbate preꢀ
decomposes to the oxidized disulfide. Under these assay
conditions, biotinꢀGSSG does not label any proteins
(Figure 5f), confirming selective conjugation of beꢀ
tween nitrosothiols and sulfinic acids is selective and biꢀ
directional.
treatment (Figure 4a). Sꢀnitrosoꢀglutathione (GSNO) is
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reported to Sꢀnitrosate GAPDH via transꢀnitrosation .
GAPDH is only labeled by biotinꢀSO H after GSNO
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addition. Labeling is reversed by incubation with dithioꢀ
threitol, which cleaves the thiosulfonate linkage (Figure
4
b). Further highꢀresolution mass spectrometry analysis
of denatured and labeled GAPDH tryptic peptides conꢀ
firmed thiosulfonate formation (Figure S9).
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Biotin-GSNO detects S-sulfinated proteins. We next
asked what would happen if we reversed our detection
scheme, by applying Sꢀnitrosothiolꢀlinked probes to deꢀ
tect endogenous Sꢀsulfination. Similar reactivity was
recently reported using an arylꢀnitroso probe for conjuꢀ
gation to sulfinic acid standards to form a stable Nꢀ
2
2
sulfonylbenzisoxazolone . While both approaches use
nitroso moieties to react with sulfinic acids, each follow
different mechanistic pathways.
Figure 5. BiotinꢀGSNO (1 mM) labels a unique profile of Sꢀ
sulfinated proteins in HEK293T cell lysates. (a) BiotinꢀGSNO
labeling is not competed by Sꢀmethylglutathione (1 mM). (b)
GSNO competes with biotinꢀGSNO for labeling native Sꢀ
sulfinated proteins. (c) BiotinꢀGSNO labeling of Sꢀsulfination is
unaffected by dimedone (1 mM). (d) Proteins first labeled with
biotinꢀGSNO are then lost after TCEP (5 mM) addition. (e)
Peroxide preꢀtreatment in iodoacetamide alkylated lysates
eliminates biotinꢀGSNO labeling, suggesting terminal oxidation
of sulfinic acids to nonꢀreactive sulfonic acids. (f) BiotinꢀGSSG, a
putative contaminant in biotinꢀGSNO, does not label any proteins.
Accordingly, biotinꢀGSNO was synthesized in one
step from biotinꢀNHS and GSNO in deꢀgassed phosꢀ
phate buffer in the dark. After HPLC purification, the
probe was stored as singleꢀuse aliquots at ꢀ80 °C to minꢀ
imize formation of oxidized biotinꢀGSSGꢀbiotin, or used
immediately for best results. Mammalian cell lysates
were first denatured in 6 M urea in PBS, and treated
with dithiothreitol (DTT) to reduce disulfides (Figure
21a, 39
The redox chaperone DJꢀ1 (PARK7) readily forms a
S10) without affecting sulfinic acids
. This is essenꢀ
21a, 21d
stable sulfinic acid at Cys106
, which is critical for
tial as to prevent exchange with any deꢀnitrosated glutaꢀ
thione in the probe stock. The reduced lysate was then
incubated with iodoacetamide to block free thiols, and
precipitated to remove any residual reactants. After solꢀ
ubilization in 6 M urea, biotinꢀGSNO was added to label
Sꢀsulfinated proteins for gelꢀbased analysis. Labeling is
not affected by pretreatment with Sꢀmethyl glutathione
21b
suppressing redox stress . DJꢀ1 also reduces nitrosative
21b, 21c, 40
stress
, suggesting that oxidized DJꢀ1 may react
directly with cellular nitrosothiols forming a thiosulꢀ
fonate, which is readily reduced by cellular thiols like
glutathione. Here, we demonstrate that under nonꢀ
denaturing oxidative conditions, DJꢀ1 reacts with Nꢀ
acetylꢀSꢀnitrosoꢀcysteine methyl ester and GSNO (Fig-
ure 6). After peroxide and iodoacetamide treatment, naꢀ
tive DJꢀ1 was incubated with NꢀacetylꢀSꢀnitrosocysteine
methyl ester and digested with trypsin for highꢀ
resolution LCꢀMS analysis. High resolution MS/MS
analysis unambiguously confirmed thiosulfonate forꢀ
mation at Cys106 (Figure S11). Furthermore, peptides
lacking a cysteine were unaffected by up to 200 mM
peroxide treatment. In the absence of peroxide, DJꢀ1
Cys106 is fully alkylated by iodoacetamide and no labelꢀ
ing with GSNO is detectable. After incubation with 10
mM peroxide, Cys106 is detected primarily as the thioꢀ
sulfonate conjugate, with partial conversion to the unreꢀ
active sulfonic acid. Oxidation is further enhanced after
treatment with 200 mM peroxide, which eliminates all
(Figure 5a), but competed by excess GSNO (Figure
5
b). Together, these controls confirm nitrosothiolꢀ
dependent labeling of iodoacetamide resistant protein
modifications with no bias towards specific sites. Simiꢀ
larly, dimedone had no effect on biotinꢀGSNO labeling,
confirming there is no cross reactivity with thiosulꢀ
fonates, sulfinic acids, or that any residual sulfenic acids
are present after denaturation (Figure 5c). Additionally,
all labeling is eliminated by postꢀincubation with TCEP,
which reduces the thiosulfonate product (Figure 5d).
Pretreatment of iodoacetamide labeled lysates with hyꢀ
drogen peroxide (50 mM) prevented any reaction with
biotinꢀGSNO, presumably by oxidizing sulfinic acids to
sulfonic acids (Figure 5e). Finally, oxidized glutathione
(GSSG) was coupled to biotinꢀNHS to examine if any
Cys106ꢀSO H and prevents thiosulfonate formation.
2
nonꢀspecific labeling occurred by disulfide exchange.
Even though disulfides are reduced and alkylated at the
beginning of the assay, incomplete alkylation could lead
to significant false positives if the biotinꢀGSNO probe
Both Cys46 and Cys53 did not react with GSNO after
peroxide treatment, as they were directly converted to
the unreactive sulfonic acid.
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After elution of tryptic peptides, resinꢀbound biotinylatꢀ
ed peptides were eluted with TCEP and alkylated with
maleimide, enabling detection of an additional 98 sites
of Sꢀnitrosation by mass spectrometry (Table S2). These
lists include nearly all previously annotated Sꢀnitrosated
proteins, including ion channels, chaperones, peroxireꢀ
doxins, p53, HDACs, hundreds of metabolic enzymes,
as well as a rich set of novel proteins.
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45
46
47
48
49
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52
53
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Figure 6. Analysis of thiosulfonate formation on recombinant
human DJꢀ1. Purified, recombinant DJꢀ1 was treated with buffer,
1
0 mM hydrogen peroxide, or 200 mM peroxide. Samples were
treated with iodoacetamide (IAM) to block free thiols, and excess
reagents were removed by gel filtration before incubating with
GSNO. The relative abundance of each of the modified peptide
was measured by mass spetrometry of trypic peptides. The
peptide abundances were normalized to reflect relative changes
within each condition. Error bars represent standard deviations of
from three replicates. The control peptide E –K showed no
Figure 7. Profiling native Sꢀnitrosation and Sꢀsulfination in mouse
tissues. (a) Sꢀnitrosation profile of mouse tissues labeled with
6
4
89
peroxideꢀdependent changes.
biotinꢀSO H. (b) Sꢀsulfination profile of mouse tissues labeled
2
Profiling native S-nitrosation and S-sulfination. Imꢀ
mortalized mammalian cell lines are adapted to atmosꢀ
pheric oxygen, potentially augmenting the profile of
oxidative modifications. Therefore, we isolated mouse
tissues for immediate processing and gelꢀbased analysis.
Surprisingly, we detect defined tissueꢀspecific patterns
of both Sꢀnitrosation (Figure 7a) and Sꢀsulfination (Fig-
ure 7b), demonstrating orthogonal protein targets of
each modification in vivo.
with biotinꢀGSNO using matched protein loading, fluorescence
detection, and image settings.
To profile Sꢀsulfination, heavy or light cell 293T cell
lysates were separately treated with DTT, alkylated with
excess iodoacetamide, and treated with either biotinꢀ
GSNO or free biotin. After enrichment and mass specꢀ
trometry analysis, nearly 300 Sꢀsulfinated proteins were
specifically enriched, including DJꢀ1 (PARK7), phosꢀ
phatases, metabolic enzymes, and a partially overlapping
set of oxidized proteins (Table S3). Resinꢀbound pepꢀ
tides were eluted using TCEP reduction, enabling identiꢀ
fication of an additional 30 specific sites of Sꢀsulfination
(Table S4), altogether providing the first largeꢀscale
analysis of this redox modification.
To annotate the endogenous targets of each redox
modification in cells, both biotinꢀSO H and biotinꢀ
2
GSNO conjugates were profiled using stableꢀisotope
labeling with amino acids in cell culture (SILAC) for
quantitative mass spectrometry proteomics. For profiling
Sꢀnitrosation, heavy or light cell 293T cell pellets were
lysed by sonication in 6 M urea, treated with excess ioꢀ
Next, to evaluate the relative occupancy of each redox
modification, we quantified both Sꢀnitrosation and Sꢀ
sulfination enrichment in comparison with relative proꢀ
tein abundance. Unenriched 293T proteomes were anaꢀ
doacetamide, and incubated with either biotinꢀSO H or
2
biotinꢀSO H. After chloroform/methanol precipitation,
3
the two proteomes were combined for streptavidin enꢀ
richment, trypsin digestion, and high resolution mass
spectrometry analysis. Through a combination of 4 bioꢀ
logical replicates, each with 2 technical replicates, a total
of 992 proteins were identified with SILAC ratios > 5
41
lyzed using labelꢀfree quantification (Table S5) based
on the integrated ion intensity of the top 3 most intense
ions for each protein. Individual protein values from the
probe enrichment were divided by their corresponding
relative abundance, providing a distribution of ratios
reflecting proportionally higher modification occupancy
(
biotinꢀSO H/biotinꢀSO H), quantified in ≥ 3 replicates,
2 3
and represented by ≥ 3 quantified peptides (Table S1).
(Figure 8a and Table S6). This analysis is critical to
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identify not just abundant proteins with fractional oxidaꢀ
tion, but to highlight proteins with greater representative
modification occupancy. Importantly, this is not an absoꢀ
lute stoichiometry. This value reflects the relative enꢀ
richment efficiency as compared to the estimated relaꢀ
tive abundance, and helps identify proteins that may be
low abundance, but are highly modified.
For both redox modifications, the majority of proteins
were observed with low ratios, signifying poor relative
enrichment and low modification stoichiometry characꢀ
teristic of abundant proteins, including several heat
shock, cytoskeletal, and ribosomal proteins. In contrast,
Sꢀnitrosated proteins with large ratios signify high relaꢀ
tive occupancy, including several metabolic enzymes
and proteins with metal coordination sites, such as
HDAC1 and carbonic anhydrase. Interestingly, Sꢀ
sulfinated proteins with large ratios include validated
oxidation prone enzymes such as peroxiredoxins, thioreꢀ
doxin, pyruvate kinase, and triosephosphate isomerase.
Approximately 175 proteins were selectively enriched
with both probes, revealing inherent preferences for each
redox modification (Figure 8b). Interestingly, DJꢀ1 was
found to be both Sꢀnitrosated and Sꢀsulfinated. Both
Cys46 and C53 are surface exposed and established sites
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of Sꢀnitrosation and the tryptic peptide containing
Cys53 was identified in our endogenous siteꢀspecific
2
1a,
analysis. In contrast, Cys106 is primarily Sꢀsulfinated
2
1b, 21d
,
demonstrating how a single endogenous protein
can harbor more than one distinct redox modifications.
Conclusions
Overall, this approach highlights proteins with enꢀ
hanced susceptibility to distinct redox modifications,
opening new opportunities for multiplexed profiling of
diseaseꢀdependent mutually competitive cysteine modiꢀ
fications. This simplified, direct approach bypasses the
hazards of mercuryꢀbased affinity reagents, and avoids
complex disulfide chemistry commonly used for ascorꢀ
bateꢀdependent reduction strategies. While the thiosulꢀ
fonate linkage is not ideal for stable enrichment, we find
that after proper alkylation of free thiols, thiosulfonates
are sufficiently stable for nonꢀreducing gelꢀbased analyꢀ
sis and mass spectrometry profiling. Importantly, these
findings establish that sulfinic acids possess intrinsic
reactivity that may contribute to cellular redox regulaꢀ
tion. As observed for human DJꢀ1, once Cys106 is oxiꢀ
dized to a sulfinic acid, it readily converts Sꢀnitrosated
thiols to an easily exchangeable thiosulfonate. Future
studies will explore if enzymes, once exposed to oxidaꢀ
tive stress, form nucleophilic sulfinic acids that aid cells
from accumulating further oxidative damage.
Figure 8. Relative comparative proteinꢀlevel occupancy of redox
modifications. (a) Histogram of calculated relative occupancy
ratios of Sꢀnitrosated proteins (left) compared to Sꢀsulfinated
proteins (right), derived from labelꢀfree quantiation. (b)
Comparative analysis of relative occupancy ratios of both S-
nitrosated and Sꢀsulfinated proteins reveals inherent preferences
towards each redox modification. Arbitrary lines and color
boundaries are presented, separating abundant, low occupancy
proteins (grey), from highly Sꢀnitrosated (blue), highly Sꢀ
sulfinated (green), and proteins with enhanced occupancy for each
modification (red).
ASSOCIATED CONTENT
Supporting Information. Additional figures, tables, comꢀ
pound characterization, and detailed methods. This material
is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
brentrm@umich.edu
ACKNOWLEDGMENT
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Support for these studies was provided by the National
Institutes of Health R00 CA151460, DP2 GM114848, T32
CA140044 Proteome Informatics of Cancer Training Grant
A.M.K), F31 EB019320 (C.T.M.B.T.), the National Sciꢀ
ence Foundation (Graduate Research Fellowship to
A.M.K.), the American Heart Association
4POST20420040 (J.D.M.), and the University of Michiꢀ
gan.
20.
Chang, T. S.; Jeong, W.; Woo, H. A.; Lee, S. M.; Park, S.;
Rhee, S. G. J. Biol. Chem. 2004, 279, 50994.
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