Profiling protein thiol oxidation in tumor cells using
sulfenic acid-specific antibodies
Young Ho Seoa and Kate S. Carrolla,b,1
aLife
Sciences Institute and bDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109-2216
Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved August 10, 2009 (received for review March 18, 2009)
R
eactive oxygen species (ROS) play important roles in normal
biological functions and pathological processes (1). Cancer
cells are frequently under persistent oxidative stress due to
oncogenic stimulation, increased metabolic activity, and mitochondrial malfunction (2, 3). Elevated ROS generation in cancer
cells serves as an endogenous source of DNA-damaging agents
that promote genetic instability (4). Mounting evidence also
supports a physiological role for ROS as second messengers in
intracellular signaling cascades that control cell growth, proliferation, migration, and apoptosis (5–7). In these pathways,
stimulation of various cell surface receptors activate the
NADPH oxidase complex to generate a burst of hydrogen
peroxide (H2O2). H2O2 modulates signal transduction through
chemoselective oxidation of cysteine residues in proteins,
thereby altering their cellular function (8–11). In cancer cells,
increased ROS may generate inappropriate proliferation signals
and thus, contribute to tumor growth and other biological events
that promote malignancy (12).
The connection between H2O2 signaling, thiol oxidation, and
enhanced signaling through growth factor pathways provides a
framework to develop therapeutics that selectively target cancer
cells based on their redox status (2, 12–14). Central to achieving
this goal is the development of tools and assays that facilitate
characterization of the molecular events associated with tumorigenesis and evaluation of patient response to therapeutic treatment. Toward this end, immunochemical methods offer a potentially powerful and facile approach for monitoring changes in
thiol oxidation state, as an indicator of normal biological function and pathological processes. Antibodies are available to
detect S-glutathionylation (Cys-SSG) (15), S-nitrosylation (CysSNO) (16), and sulfonated cysteine (Cys-SO3⫺) (17). However,
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0903015106
Results and Discussion
Generation of 2-Thiodimedone-Specific IgG. To detect H2O2-
mediated thiol oxidation, we envisioned an antibody that could
specifically recognize cysteine sulfenic acid. However, the structures of related oxoforms, such as sulfinic or sulfonic acid, are
very similar, and thus, isolation of an antibody devoid of
cross-reactivity might be difficult. In addition, the inherent
reactivity of sulfenic acids could make it challenging to generate
a homogenous hapten-protein conjugate. To address these issues, we have taken a chemical approach to convert the sulfenic
acid into an epitope that enables antibody recognition (Fig. 1).
Dimedone is a cell-permeable, nucleophilic reagent that reacts
selectively with the electrophilic sulfur atom in sulfenic acid to
form a stable thioether product (Fig. 1 A) (18, 19). Since other
functional groups in proteins do not react with dimedone, this
chemical tag provides a unique starting point from which to
differentiate sulfenic acids from all other cysteine oxoforms.
Based on this strategy, we designed and synthesized the hapten
depicted in Fig. 1B, which consists of the dimedone moiety on a
cysteamine backbone with a five-carbon linker to keyhole limpet
hemocyanin (KLH). The resulting hapten-protein conjugate was
then used to elicit rabbit ␣-hapten-Ig gamma (IgG). In vitro
experiments show that the antibodies only recognized the oxidized form of glyceraldehyde 3-phosphate dehydrogenase
(GAPDH, Fig. 1C) and papain (Fig. S1 A) when modified by
dimedone, verifying that the antibodies discriminate the adduct
from other protein functional groups and that recognition is
independent of protein sequence. In addition, the ␣-hapten
antibody displays greater sensitivity in Western blot analysis
compared to other chemical methods of sulfenic acid detection
Author contributions: Y.H.S. and K.S.C. designed research; Y.H.S. performed research;
Y.H.S. and K.S.C. analyzed data; and Y.H.S. and K.S.C. wrote the paper.
Conflict of interest statement: The University of Michigan is presently negotiating with
Assay Designs to commercialize this antibody along with the necessary reagents for
labeling protein sulfenic acids.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To
whom correspondence should be addressed. E-mail: katesc@umich.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0903015106/DCSupplemental.
PNAS 兩 September 22, 2009 兩 vol. 106 兩 no. 38 兩 16163–16168
CHEMISTRY
cancer 兩 chemical biology 兩 redox signaling 兩 thiol modification
these reagents have limited utility in monitoring protein oxidation that occurs in H2O2 signaling because (i) disulfide bonds
undergo thiol-disulfide exchange and can easily be reduced,
leading to a loss signal, (ii) S-nitrosylation is not mediated by
H2O2, and (iii) ␣-sulfonylated cysteine antibodies only recognize
specific peptide sequences. Here we present the development
and application of a method for detecting cysteine sulfenic acid,
the initial oxidation product that results when a thiolate reacts
with H2O2. We show that this antibody exhibits high specificity
and sensitivity for sulfenic acids in proteins, independently of
sequence content. Application of this immunochemical approach, to protein lysate arrays and cancer cell lines, allowed us
to monitor changes in thiol redox status and revealed diversity
in sulfenic acid modifications among different subtypes of breast
tumors.
BIOCHEMISTRY
Hydrogen peroxide (H2O2) functions as a second messenger that
can activate cell proliferation through chemoselective oxidation of
cysteine residues in signaling proteins. The connection between
H2O2 signaling, thiol oxidation, and activation of growth pathways
has emerged as fertile ground for the development of strategies
for cancer treatment. Central to achieving this goal is the development of tools and assays that facilitate characterization of the
molecular events associated with tumorigenesis and evaluation of
patient response to therapy. Here we report on the development
of an immunochemical method for detecting sulfenic acid, the
initial oxidation product that results when a thiolate reacts with
H2O2. For this approach, the sulfenic acid is derivatized with a
chemical tag to generate a unique epitope for recognition. The
elicited antibody is exquisitely specific, context-independent, and
capable of visualizing sulfenic acid formation in cells. Applying this
approach to several systems, including cancer cell lines, shows it
can be used to monitor differences in thiol redox status and reveals
a diverse pattern of sulfenic acid modifications across different
subtypes of breast tumors. These studies demonstrate a general
strategy for producing antibodies against a specific oxidation state
of cysteine and show the utility of these reagents for profiling thiol
oxidation associated with pathological conditions such as breast
cancer.
A
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Fig. 1.
Strategy for tagging and antibody-based detection of protein
sulfenic acids. (A) Chemoselective alkylation of sulfenic acids by dimedone
yields the synthetic epitope, which is recognized by the ␣-hapten antibody. (B)
Structure of the hapten conjugate used to elicit sulfenic acid-specific antibodies. (C) GAPDH was incubated with combinations of hydrogen peroxide and
dimedone as indicated; reaction products were analyzed by Western blot with
the ␣-hapten antibody.
such as dimedone-biotin (20, 21) and an azide-tagged analog of
dimedone (22, 23) (Fig. S1B).
Detecting Thiol Oxidation in Cells. With in vitro results showing the
specificity of the ␣-hapten antibody in hand, we next tested the
ability of this reagent to image sulfenic acid in biological samples
using immunofluorescence (IF) staining and fluorescence microscopy (Fig. 2A). Live human cervical cancer (HeLa) cells
were incubated with dimedone to trap and tag protein sulfenic
acids. In subsequent steps, cells were washed, fixed, permeabilized for antibody accessibility, and then probed with the ␣-hapten
antibody. IF staining for sulfenic acids was diffuse throughout the
cytoplasm of HeLa cells (Fig. 2 A, top); perinuclear and peripheral staining of a lace-like network, reminiscent of the ER
network, was also revealed (Fig. 2 A, middle). Control experiments with cells excluding dimedone showed no background
fluorescence (Fig. 2 A, bottom), indicating that the observed IF
staining was specific for dimedone-tagged sulfenic acids. Moreover, trypan blue exclusion after dimedone incubation confirms
that cells remained viable throughout the treatment (Fig. S2 A).
Next, we investigated whether dynamic changes in sulfenic acid
formation could be observed using the fluorescence microscopy
and the ␣-hapten antibody. To simulate acute oxidative stress,
HeLa cells were exposed to hydrogen peroxide. At various time
points after oxidation, cells were incubated briefly with dimedone, and then processed for immunofluorescence staining as
described above. Fig. 2B shows that IF staining for sulfenic acids
increased in the first 15 min following stimulation with hydrogen
peroxide and diminished to an undetectable level within 30 min.
To verify detection of protein sulfenic acid modifications, we
performed Western blot analysis with lysates derived from cells
incubated with dimedone (Fig. 2C). HeLa cells that were not
exposed to dimedone showed some nonspecific immunoreactivity (Fig. 2C, lane 1, asterisks; see also Fig. 5). This background
signal most likely arises from cross-reactivity of antibodies
against the carrier protein, KLH, with a small number of
unknown proteins present in human cells. Nonetheless, there
16164 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0903015106
B
0 min
5 min
15 min
30 min
Dimedone + H2O2
α-GAPDH
Fig. 2. Immunofluorescence microscopy and Western blot showing sulfenic
acid-modified proteins in HeLa cells. (A) HeLa cells were incubated in media
containing 5 mM dimedone (top and middle) or DMSO (2.5% vol/vol) (bottom)
for 1 h at 37 °C and analyzed for protein sulfenic acids (red). Nuclei were
counterstained with DAPI (blue). (Scale bars, 60 ␮m.) (B) HeLa cells were
untreated or treated with 1 mM H2O2 for 5, 15, or 30 min at 37 °C. At the end
of each time point, cells were washed and incubated in media containing 5
mM dimedone for 15 min at 37 °C and then analyzed for protein sulfenic acids
(red). Nuclei were counterstained with DAPI (blue). (C) Protein was isolated
from cells incubated in media containing 2.5, 5, or 10 mM dimedone or DMSO
(2.5% vol/vol) for 2 h at 37 °C. Sulfenic acid-modified proteins were analyzed
by Western blot with the ␣-hapten antibody. Arrowhead, protein bands
corresponding to sulfenic acid-modified actin, GAPDH and PrxI (see also SI
Materials and Methods); asterisk, immunoreactive background band.
was a clear, dose-dependent increase in the extent of protein
labeling for dimedone-treated cells (Fig. 2C, lanes 2–4). Using
protein-specific antibodies (see SI Materials and Methods), we
identified three proteins in our Western blot analysis as actin,
GAPDH, and peroxiredoxin I (PrxI), denoted by arrowheads in
Fig. 2C. Actin and GAPDH are highly abundant proteins and
known to contain reactive cysteines that are readily oxidized to
sulfenic acid (24, 25). PrxI is an antioxidant protein that uses
cysteine as the primary site of oxidation during the reduction of
H2O2 (26). Positive identification of these sulfenic acid-modified
proteins confirms that our methodology is capable of detecting
H2O2-mediated thiol oxidation in cells. Furthermore, time and
dose-dependent protein labeling was also observed in Jurkat
T-cells, demonstrating the generality of the method (Fig. S2 B
and C). Taken together, the IF and Western blot results establish
that the ␣-hapten antibody is capable of detecting sulfenic
acid-modified proteins, which have been trapped and tagged
with dimedone in cells.
Protein Microarray-Based Approach to Profile Thiol Oxidation. Pro-
tein microarrays are used as a high-throughput method to
identify protein-protein interactions, kinase substrates, targets
of biologically active small molecules, and profile posttranslational modifications such as phosphorylation (27, 28). To
the best of our knowledge, however, this approach has not been
applied to investigate protein thiol oxidation. To explore the
Seo and Carroll
Profiling Sulfenic Acid Levels in Tumor and Normal Breast Tissue
Lysates Using Protein Microarrays. The preceding experiments led
us to expand our study of sulfenic acid modifications in cancer
to additional tissues, and we were specifically interested in breast
carcinoma because oxidative stress is well known to modulate
estrogen receptor (ER) pathways (27–30). The array chip contained 40 pairs of surgical samples (tumor and adjacent normal
tissue) obtained from patients with ductal breast cancer, assigned to histological grades I–III and ranging in age from 35 to
85. Of these, 38 samples yielded a standard deviation of 15% or
less and were included in subsequent comparisons. Fig. 4 shows
a representative data set from these experiments and expresses
the level of thiol oxidation in each sample as the ratio of sulfenic
acid in breast tumor to the patient-matched normal according to
age (Fig. 4A) or grade (Fig. 4B). A ratio greater than 1 indicates
that thiol oxidation was elevated in the malignant tissue, relative
to the paired sample.
Well-established markers for breast cancer such as carcinomaSeo and Carroll
A
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Normal tissue
Bladder
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Breast, Colon
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Ovary, Bladder
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Squamous cell
Skin, Jurkat
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CHEMISTRY
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BIOCHEMISTRY
Fluorescence (a.u. x 107)
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utility of the protein microarray platform to profile sulfenic acid
levels, we first tested whether the ␣-hapten antibody could detect
thiol oxidation on surface-immobilized GAPDH. Protein was
applied directly to PVDF as a dot. In subsequent steps, the
membrane was treated with H2O2 and dimedone to form and
trap the sulfenic acid modification on GAPDH. The ␣-hapten
antibody recognized only oxidized GAPDH when modified by
dimedone (Fig. S3), analogous to data obtained for GAPDH in
solution (Fig. 1C).
Having established that our immunochemical approach could
be applied to a surface-immobilized substrate we then examined
sulfenic acid levels in protein microarrays using the strategy
outlined in Fig. 3A. In initial tests, we analyzed H2O2-mediated
thiol oxidation using an array chip consisting of individual tissue
lysate samples from 14 human organs, four mammalian cell lines
as well as bladder tumors and matching normal tissue from nine
individuals. After dimedone treatment, the total sulfenic acid
level in each sample was assessed using the ␣-hapten antibody
and chemifluorescence detection (Fig. 3B). Among the organs
screened, the highest level of sulfenic acid was identified in the
ovaries (Fig. 3C), where ROS are essential for several biological
processes related to reproduction, including folliculogenesis
(29). Sulfenic acid levels were also higher than the mean average
in cervix, kidney, thyroid, colon, and liver tissues (Fig. 3C). BSA
and buffer samples arrayed on the chip showed no background
signal, as expected (Fig. 3B). The cell culture process is known
to increase cellular ROS formation (1), and consistent this
general observation, elevated levels of sulfenic acid were observed in all four mammalian cell lines (Fig. 3C).
Sulfenic acid modifications have not been investigated in
malignant tissue samples and thus, we conducted a pilot study of
thiol oxidation in individuals with bladder cancer. Among the
nine patient tumor tissue samples that we examined, sulfenic
acid abundance varied by up to seven-fold (Fig. S4). In addition,
six of seven individuals with transitional or squamous cell
carcinoma exhibited a significant increase in the extent of
sulfenic acid modifications, relative to matched normal tissue
(P ⬍ 0.005 by paired t test; Fig. S4); adeno- and epidermoid
bladder carcinomas samples were also associated with elevated
levels of sulfenic acid (P ⬍ 0.001 by paired t test; Fig. S4).
Although the number of paired samples on the array chip is too
small to draw broad conclusions, these initial observations
suggest that elevated levels of sulfenic acid might be hallmark of
bladder tumor tissues. Consistent with this hypothesis, lower
total thiol groups have been reported in the blood plasma of
patients with bladder cancer, as compared to healthy controls
(26). The current data support and extend this previous study
through analysis of sulfenic acids in bladder tumor and matched
normal tissue samples.
Normal tissue
Cell culture
Fig. 3. Detecting thiol oxidation using the protein microarray platform. (A)
Strategy for detecting sulfenic acids on protein microarrays with the ␣-hapten
antibody. (B) Profiling protein sulfenic acid levels in tissue lysates. The protein
microarray was incubated in buffer containing 2 mM dimedone for 1 h and
probed for sulfenic acids (red) with the ␣-hapten antibody. Negative (BSA and
lysis buffer) and IgG (rabbit IgG spotted at 1.0 and 0.1 mg/mL) controls are also
included on the array. (C) Analysis of sulfenic acid content in human tissues
and mammalian cell lines. Chemifluorescence intensity of microarray spots is
indicated with bars. Error bars, the standard deviation of three replicate spots.
associated antigen (CA15–3, also known as MUC1) and carcinoembryonic antigen (CEA) show 1.5- to 2-fold changes in
abundance among breast cancer patients (30–32). By comparison, the sulfenic acid ratio among patients analyzed in this study
ranged from 0.25- to 4-fold (Fig. 4). When comparing two age
groups (35–49 years, n ⫽ 20 and 50–85 years, n ⫽ 18), the
younger population had three times as many individuals with a
sulfenic acid ratio greater than 1.2 (Fig. 4A). The lack of positive
correlation between sulfenic acid ratio and age in these experiments suggests that not all cancers arising with age will exhibit
PNAS 兩 September 22, 2009 兩 vol. 106 兩 no. 38 兩 16165
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Fig. 4. Comparison of sulfenic acid levels by protein microarrays from tumor
and normal breast tissue lysates. The level of oxidation in each sample expressed as the ratio of sulfenic acid in breast tumor to the patient-matched
normal and analyzed according to age (A) or histological grade (B). A ratio
greater than one indicates that thiol oxidation is elevated in malignant tissue,
relative to the paired sample. Chemifluorescence intensity of microarray spots
is indicated with bars. The standard deviation for three replicate spots was less
than ⫾ 15%.
excess levels of oxidative stress, relative to younger individuals.
This hypothesis is supported by a recent report which indicates
that expression of oxidant-responsive genes is more evident in
breast cancers diagnosed at an early age, relative to those
diagnosed at an older age (33). Among different tumor grades,
most striking is the observation that 53% of individuals with
grade II breast carcinoma (n ⫽ 17) had sulfenic acid ratios
greater than 1.0, relative to 25% in grade III (n ⫽ 16) and none
in grade I (n ⫽ 5) (Fig. 4B). Our observation that mid-stage
breast cancer is associated with higher sulfenic acid ratios is
notable and may indicate that thiol oxidation promotes tumor16166 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0903015106
Fig. 5. Profiling sulfenic acid modifications in breast cancer cells. (A) Comparative sulfenic acid analysis of matched breast cell lines HS578Bst (normal)
and HS578T (carcinoma). HS578Bst and HS578T cells were cultured in media
containing 5 mM dimedone or DMSO (2.5% vol/vol) for 2 h at 37 °C. After
washing with PBS, cells were harvested in lysis buffer and sulfenic acidmodified proteins were analyzed by Western blot with the ␣-hapten antibody.
Equal protein loading was verified by ␣-actin (bottom). Asterisks denote a
background immunoreactive band. (B) Profiling sulfenic acid-modified proteins in breast cancer cell lines. BT20, BT474, MCF7, MDA-MB231, and MDAMB468 cell lines were cultured in media containing 5 mM dimedone or DMSO
(2.5% vol/vol) for 3 h at 37 °C. After washing with PBS, cells were harvested in
lysis buffer and sulfenic acid-modified proteins were analyzed by Western blot
using the ␣-hapten antibody. Equal protein loading was verified by ␣-actin
(bottom). Asterisks denote a background immunoreactive band.
igenesis among a subset of patients, possibly by activating
proteins that function in growth factor signaling pathways. To
further validate this hypothesis, additional studies will be
required.
Comparative Sulfenic Acid Profiling of Human Breast Cancer Cell Lines.
Although many important insights into thiol redox modifications
have been gained through in vitro studies (34–38), proteins are
subject to oxidation upon cell lysis (39, 40). Therefore, we
extended our protein microarray analysis by profiling sulfenic
acid modifications directly in human breast cell lines (Fig. 5). For
these experiments, protein oxidation was characterized by Western blot analysis of whole-cell lysates derived from cells treated
Seo and Carroll
Materials and Methods
Antibody Generation. KLH (Keyhole Limpet Hemocyanin) protein was activated
by adding sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1carboxylate), a hetero-bifunctional crosslinker containing an amine-reactive Nhydroxysuccinimide and a sulfhydryl-reactive maleimide group. The hapten (synthesized as described in SI Appendix and Fig. S5) was conjugated to succinylated
KLH overnight at pH 8 and the product was then purified over a PD10 gel
filtration column (GE Healthcare). Two New Zealand White (specific pathogenfree grade) rabbits were immunized subcutaneously with conjugate in a 50:50
emulisification with adjuvant [either Freunds complete (FCA) or Freunds incomplete (FIA)] according to the following schedule: day 0 boost (FCA), day 14 boost
(FIA), and day 28 boost (FIA). The rabbits were bled on days 35 and 40 after the
primary boost, the red blood cells spun out by centrifugation, and the remaining
antisera used for testing, without further purification.
Dimedone Labeling of GAPDH and Papain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit muscle was obtained as a powder (Sigma).
GAPDH (4 mg/mL in PBS pH 7.4) was treated with 1 mM DTT for 20 min at 0 °C.
After reduction, DTT was removed by ultrafiltration using an Amicon Ultra-4
10 KDa MWCO centrifugal filter unit (Millipore). Papain was obtained as a
powder (Sigma) and further purified as previously described (22). Stocks of
dimedone used in labeling studies were made up in DMSO-0.1 M Bis-Tris䡠HCl,
pH 7.4 (1:1). To obtain dimedone-tagged GAPDH or papain, protein (10 ␮M in
PBS) was treated with 5 mM dimedone or DMSO (2.5% vol/vol) followed by 0,
10, 20, or 60 ␮M H2O2 for 30 min at room temperature. The reaction was
terminated by adding 10% ␤-mercaptoethanol in XT sample buffer (Bio-Rad)
and analyzed by Western blot using the ␣-hapten antibody.
Seo and Carroll
Fluorescence Microscopy. HeLa cells (⬃2.0 ⫻ 105) were seeded on collagencoated coverslips in DMEM media supplemented with 10% FBS, 1% PSG, and
1⫻ NEAA 16 h before analysis. Cells were incubated with 5 mM dimedone or
DMSO (2.5% vol/vol) for 1 h at 37 °C in DMEM media (2% FBS, 1% PSG, and 1⫻
NEAA). Next, cells were washed with PBS (2 ⫻ 5 min) and fixed with acetonemethanol (1:1) for 10 min at ⫺20 °C. After fixation, cells were washed with PBS
(3 ⫻ 5 min), blocked with 10% FBS in PBS for 30 min at room tempertature,
washed (2 ⫻ 10 min) with DMEM media (2% FBS, 1% PSG, and 1⫻ NEAA) and
then with PBS (2 ⫻ 10 min). Cells were incubated with the ␣-hapten antibody
in PBST (1:3,000) for 30 min, washed with PBS (3 ⫻ 10 min) and incubated with
␣-rabbit Alexa Fluor 594 (Invitrogen, 1:1,000) in PBST for 1 h at room temperature. After washing with 5% FBS (2 ⫻ 10 min) and PBS (2 ⫻ 10 min) slides were
stained with 0.1 mg/mL DAPI for 1 min, rinsed with PBS, mounted with
Fluoromount G (Southern Biotech) and analyzed using an Olympus BX-51
microscope. To monitor dynamic changes in sulfenic acid formation, HeLa cells
were untreated or treated with 1 mM H2O2 for 5, 15, or 30 min at 37 °C. At the
end of each time point, cells were washed and cultured in fresh media
containing 5 mM dimedone for 15 min at 37 °C. After dimedone treatment,
cell samples were processed for fluorescence microscopy, as described above.
Detecting Protein Sulfenic Acids in Cultured Cells. HeLa cells were trypsinized,
pelleted (200 ⫻ g at room temperature) and resuspended in DMEM containing
2% FBS, 1% PSG, and 1⫻ NEAA. Cells were then treated with 2.5, 5, or 10 mM
dimedone or DMSO (2.5% vol/vol) for 2 h at 37 °C. Breast cell lines were
processed as above, but resuspended in DMEM containing 0.5% FBS, 1% PSG
and then treated with 5 mM dimedone or DMSO (2.5% vol/vol) for 3 h at 37 °C.
After incubation, cells were harvested, washed in PBS and then resuspended
in 100 ␮L ice-cold lysis buffer (1.0% Nonidet P-40, 50 mM Tris-HCl, pH 8, 150
mM NaCl, and protease inhibitors) and incubated for 30 min on ice with
frequent mixing. The supernatant was collected by centrifugation (20,000 ⫻
g at 4 °C) and the protein concentration of the resulting lysate was determined
by Bradford assay (Bio-Rad). Sulfenic acid-modified proteins were analyzed by
Western blot using the ␣-hapten antibody.
Western Blot. Proteins were resolved by SDS/PAGE using Criterion XT 4 –12%
Bis-Tris gels (Bio-Rad) in XT Mes running buffer, transferred to PVDF membrane and then blocked with 5% nonfat dried milk in TBST overnight at 4 °C
or for 1 h at room temperature. The membrane was then washed in TBST (2 ⫻
10 min) and incubated with ␣-hapten antibody (1:2,000 –1:4,000) for 1 h at
room temperature. Next, the membrane was washed with TBST (3 ⫻ 10 min)
and incubated with goat ␣-rabbit-HRP in TBST (1:5,000 –10,000) for 30 min at
room temperature. After washing with TBST (3 ⫻ 10 min), the blot was
developed using ECL Plus detection reagent (GE Healthcare) and the chemifluorescent signal was visualized using a Typhoon scanner (GE Healthcare). To
demonstrate equal protein loading streptavidin-HRP blots were inactivated
with 15% H2O2 in TBST and re-probed with ␣-GAPDH or ␣-actin (Santa Cruz
Biotechnology), followed by rabbit ␣-mouse-HRP.
Tissue Lysate Protein Microarray. SomaPlex reverse phase protein microarrays
with cell culture and tissue lysate samples were obtained from Protein Biotechnologies (see also SI Materials and Methods). Microarray slides were
washed with water, TBST (2 ⫻ 5 min) and incubated with 2 mM dimedone in
TBST for 15 min at room temperature. After washing with TBST (3 ⫻ 5 min), the
slide was incubated in ZeptoBlock (Protein Biotechnologies) overnight at 4 °C
and washed with TBST (5 ⫻ 5 min). The slide was then incubated with ␣-hapten
antibody in TBST (1:5,000) for 1 h at room temperature, washed with TBST (6 ⫻
5 min) and incubated with goat ␣-rabbit-HRP (1:10,000) for 1 h at room
temperature. After washing in TBST (6 ⫻ 5 min), the slide was developed with
ECL plus detection reagent and the chemifluorescent signal was visualized
using a Typhoon scanner. The resulting signal intensities were quantified
using ImageQuant software.
ACKNOWLEDGMENTS. This work was supported by the Life Sciences Institute,
Leukemia and Lymphoma Society Special Fellows Award 3100-07, and the American Heart Association Scientist Development Grant 0835419N (to K.S.C.).
PNAS 兩 September 22, 2009 兩 vol. 106 兩 no. 38 兩 16167
CHEMISTRY
Conclusion
Understanding the complexities of protein thiol oxidation in normal function and disease states remains a major challenge. Here we
have described a powerful method for detection of sulfenic acids
that expands the arsenal of tools for analyzing thiol oxidation in
cells. The use of antibodies that target a specific cysteine oxidation
state capitalizes on the sensitivity and selectivity afforded by
immunochemical methodology, combined with relatively simple
preparation and potentially broad applications. A key feature of this
approach is chemical functionalization of the sulfenic acid, which
converts the modification into an epitope that can be recognized by
the ␣-hapten antibody and facilitates discrimination over similar
modifications, particularly sulfinic and sulfonic acid. Applying this
approach to several systems, including human cancer cell lines,
shows it can be used to monitor differences in thiol redox status and
reveals a distinct pattern of sulfenic acid modifications across breast
tumor subtypes. Together, these studies demonstrate a general
strategy for producing antibodies against a specific oxidation state
of cysteine and highlight the utility of this approach for profiling
thiol oxidation associated with pathological conditions such as
breast cancer.
Cell Culture. HeLa, Hs578Bst, Hs578T, MCF-7, BT-20, BT-474, MDA-MB-231, and
MDA-MB-468 cell lines were obtained from American Type Culture Collection.
HeLa, Hs578Bst, Hs578T, MCF-7, and BT-20 cell lines were grown in DMEM
media supplemented 10% FBS, 1% penicillin-streptomycin-L-glutamate (PSG)
and 1⫻ non-essential amino acids (NEAA) at 37 °C in a humidified atmosphere
of 5% CO2. BT-474 cells were grown in RPMI supplemented with 10% FBS and
1% PSG at 37 °C in a humidified atmosphere of 5% CO2. MDA-MB-231 and
MDA-MB-468 cell lines were grown in Leibovitz’s L-15 media supplemented
with 10% FBS and 1% PSG at 37 °C.
BIOCHEMISTRY
with dimedone, analogous to earlier experiments in HeLa cells
(Fig. 3). Initially, we compared thiol oxidation in cell lines
derived from primary invasive ductal carcinoma (Hs578T) and
normal adjacent tissue (Hs578Bst). Fig. 5A shows that protein
sulfenic acid modifications are more prevalent in the Hs578T cell
line, relative to its normal counterpart. Next, we profiled protein
sulfenic acid modifications across cell lines that represent distinct subtypes of breast cancer, including BT20, BT474, MCF7,
MDAMB231, and MDAMB468 (Fig. 5B) [see Table S1 for
source, clinical, and pathological features of tumors used to
derive breast cancer cell lines used in this study (41)]. Each breast
cancer cell line showed a unique pattern and intensity of protein
labeling. Of particular note, the extremely invasive MDAMB231
cell line exhibited the highest level of sulfenic acid modifications.
The HER2/neu-overexpressing cell line, BT474 also exhibited
intense labeling and was further distinguished by prominent
protein bands migrating between 50–100 kDa. Experiments are
currently underway to determine the identity of sulfenic acidmodified proteins in breast cancer cell lines and will be reported
in due course.
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Seo and Carroll
SI Appendix
Chemical Synthesis of Hapten. 6-Acetylthiohexanoic acid (6) and pentafluorophenyl
trifluoroacetate (7, TFAPfp) were purchased from Aldrich. 2-bromodimedone (2) (2) and NFmoc-2-aminoethanethiol (3) (3) were synthesized as previously described.
O
FmocHN
S
O
To a solution of 2-bromodimedone (0.146 g, 0.669 mmol) in THF (2 mL) was added N-Fmoc-2aminoethanethiol (0.3 g, 1.0 mmol) followed by NaHCO3 (56 mg, 0.669 mmol) at rt. The
reaction was stirred for 2 h at rt. The reaction mixture was diluted with ethyl acetate (40 mL),
washed with water (20 mL), dried over MgSO4, concentrated in vacuo and purified by silica gel
chromatography, eluting with ethyl acetate to 9:1 ethyl acetate: methanol to provide the title
compound ( 0.204 g, 0.467 mmol) in 70% yield. Rf: 0.32 (ethyl acetate). 1H NMR (CDCl3, 300
MHz): δ 7.79 (d, J = 7.2, 2H), 7.66 (d, J = 7.2, 2H), 7.43 (t, J = 7.0, 2H), 7.34 (t, J = 7.0, 2H),
5.92 (s, 1H), 4.43 (d, J = 7.5, 2H), 4.27 (t, J = 7.2, 1H), 3.32 (q, J = 5.4, 2H), 2.69 (t, J = 5.4,
2H), 2.48 (s, 4H), 1.12 (s, 6H). ESI-LRMS calcd. For C25H27NNaO4S (M+Na+) 460.2, found
460.2.
O
H 2N
S
O
To a solution of compound 4 (15 mg, 0.034 mmol) in DMF (1 mL) was added piperidine (0.2
mL) at rt. The reaction was stirred for 20 min at rt. The reaction mixture was directly loaded to
silica gel chromatography and purified, eluting with ethyl acetate to 1:1 ethyl acetate: methanol
to provide the title compound (25 mg, 0.012 mmol) in 35% yield. Rf: 0.11 (3:1 ethyl acetate:
methanol). 1H NMR (CD3OD, 400 MHz): δ 2.93 (t, J = 6.0, 2H), 2.45 (t, J = 6.0, 2H), 2.46 (s,
4H), 1.06 (s, 6H). ESI-LRMS calcd. For C10H17NNaO2S (M+H+) 238.1, found 238.1.
O
F
O
S
O
F
F
F
F
To a solution of 6-acetylthiohexanoic acid (0.276 g, 1.45 mmol) in CH2Cl2 (10 mL) was added
pyridine (0.130 mL, 1.60 mmol) followed by TFAPfp (0.30 mL, 1.74 mmol) at rt. After being
stirred for 30 min ar rt, the reaction mixture was diluted with CH2Cl2 (50 mL) and washed with
sat. NaHCO3. The organic layer was dried over Na2SO4, concentrated in vacuo at rt and purified
by silica gel chromatography, eluting with hexanes to 1:9 ethyl acetate: hexanes to provide the
title compound (0.418 g, 1.17 mmol) in 81% yield. Rf: 0.55 (1:9 ethyl acetate: hexanes). 1H
NMR (CDCl3, 300 MHz): δ 2.89 (t, J = 7.2, 2H), 2.67 (t, J = 7.2, 2H), 2.33 (s, 3H), 1.90-1.74 (m,
2H), 1.70-1.58 (m, 2H), 1.54-1.45 (m, 2H).
1
O
O
H
N
S
S
O
O
To a solution of compound 8 (43 mg, 0.120 mmol) in DMF (2 mL) was added N,Ndiisopropylethylamine (25 μL, 0.144 mmol) followed by amine 5 (31 μL, 0.144 mmol) at rt
under argon. The reaction mixture was warmed to 50 °C. After being stirred for 1.5 h at 50 °C,
compound 8 (43 mg, 0.120 mmol) in DMF (2 mL) was added. The reaction was stirred for 2.5
more hours at 50 °C under argon. The reaction mixture was concentrated in vacuo at 30 °C,
diluted with CH2Cl2 (80 mL), washed with H2O (80 mL), dried over Na2SO4, concentrated in
vacuo at rt and purified by silica gel chromatography, eluting with ethyl acetate to 5:1 ethyl
acetate: methanol to provide the title compound (41 mg, 0.106 mmol) in 88% yield. Rf: 0.29
(3:1 ethyl acetate: methanol). 1H NMR (acetone-d6, 400 MHz): δ 7.12 (s, 1H), 3.30 (q, J = 4.5,
2H), 2.84 (t, J = 6.9, 2H), 2.60 (t, J = 4.5, 2H), 2.43 (s, 4H), 2.30 (s, 3H), 2.25 (t, J = 6.9, 2H),
2.30 (s, 3H), 1.69-1.52 (m, 5H), 1.48-1.35 (m, 2H) , 1.07 (s, 6H). ESI-LRMS calcd. For
C18H29NNaO4S2 (M+Na+) 410.1, found 410.1.
O
H
N
HS
S
O
O
To a solution of compound 9 (64 mg, 0.165 mmol) in THF (2 mL) was added 1 N NaOH (2 mL)
at rt. After being stirred for 2 h at rt, the reaction was neutralized with 1 N HCl, then diluted
with CH2Cl2 (100 mL), washed with brine (50 mL), dried over Na2SO4 and concentrated in
vacuo. The crude mixture was incubated with DTT (40 mg) in 1 mL of PBS (pH 8) and 1 mL of
MeCN for 1 h at rt and then purified by C18 reverse-phase HPLC (10 μm, 21.2×150 mm,
Beckman Coulter) with a gradient of 0% B to 50 % B in 50 min (buffer A: 0.1% TFA in H2O;
buffer B: 0.1% TFA in CH3CN) at a flow rate of 10 mL/min to provide the title compound (39
mg, 0.113 mmol) in 68% yield. 1H NMR (acetone-d6, 400 MHz): δ 7.61 (s, 1H), 3.28 (q, J = 6.0,
2H), 2.57 (t, J = 6.0, 2H), 2.51 (q, J = 7.2, 2H), 2.42 (s, 4H), 2.24 (t, J = 7.2, 2H), 1.69-1.58 (m,
5H), 1.48-1.40 (m, 2H) , 1.07 (s, 6H). 13C NMR (acetone-d6, 100 MHz): δ174.62, 106.21, 39.97,
36.91, 36.78, 34.60, 31.98, 30.50, 28.58, 28.32, 25.91, 24.77. ESI-LRMS calcd. For
C16H28NO3S2 (M+H+) 346.2, found 346.2.
2
Supporting Information
Seo and Carroll 10.1073/pnas.0903015106
SI Materials and Methods
General Experimental. Unless otherwise noted, all reactions were
performed under an argon atmosphere in oven-dried glassware.
All purchased materials were used without further purification.
Thin layer chromatography (TLC) was carried out using Analtech Uniplate silica gel plates. TLC plates were visualized using
a combination of UV, p-anisaldehyde, ceric ammonium molybdate, ninhydrin, and potassium permanganate staining. Flash
chromatography was performed following the method of Still et
al. (1), using Sorbent Technologies Incorporated silica gel
(32–63 ␮M, 60-Å pore size).
NMR spectra were obtained on a Varian Inova 400 (400 MHz
for 1H; 100 MHz for 13C), or a Varian Mercury 300 (300 MHz
for 1H; 75 MHz for 13C NMR) spectrometer. 1H and 13C NMR
chemical shifts are reported in parts per million (ppm) relative
to TMS, with the residual solvent peak used as an internal
reference. Low-resolution electrospray ionization (ESI) mass
spectra were obtained on a Water-Micromass LCT spectrometer
at the University of Michigan Mass Spectrometry Laboratory.
Reverse-phase HPLC purifications were performed on a Beckman Coulter System Gold 126P equipped with System Gold 166P
detector (␭ ⫽ 220) using a C18 (21.2 ⫻ 150 mm) Beckman
Coulter Ultraprep with a gradient of 0.1% TFA in H2O and
CH3CN as the mobile phase.
DAz-1, DAz-1-biotin and p-biotin were synthesized as previously
described (4). GAPDH (4 mg/mL in PBS, pH 7.4) was treated
with 1 mM DTT for 20 min. After reduction, DTT was removed
by ultrafiltration using an Amicon Ultra-4 10 KDa MWCO
centrifugal filter unit (Millipore). Stocks of dimedone, DAz-1,
and DAz-1-biotin used in labeling studies were made up in
DMSO-0.1 M Bis-Tris䡠HCl, pH 7.4 (1:1). GAPDH (5 ␮M in
PBS) was treated with 500 ␮M dimedone, 500 ␮M DAz-1-biotin,
500 ␮M DAz-1, or DMSO (2.5% vol/vol) followed by H2O2 (0,
2.5, or 5 ␮M) and incubated for 30 min at rt. DAz-1 treated
GAPDH was further incubated with 250 ␮M p-biotin for 2 h at
37 °C. Proteins were resolved by SDS/PAGE using Criterion XT
4–12% Bis-Tris gels (Bio-Rad) in XT Mes running buffer,
transferred to PVDF membrane, and then blocked with 5%
nonfat dried milk in PBST overnight at 4 °C or for 1 h at room
temperature. The membrane was then washed in PBST (2 ⫻ 10
min) and probed with ␣-hapten antibody (1:200) or HRPstreptavidin (1:100,000) for 1 h at room temperature. The
membrane was then washed with PBST (3 ⫻ 10 min) and
incubated with goat ␣-rabbit-HRP (1:100,000) for 1 h at room
temperature, washed with PBST (3 ⫻ 10 min), and developed
using ECL Plus detection reagent (GE Healthcare). Equal
protein loading was verified by probing with ␣-GAPDH.
Chemical Synthesis of Hapten. See SI Appendix.
Viability Assay of Cultured Cells. Jurkat cells were cultured as
Cell Culture. Jurkat cells were cultured in RPMI media supple-
mented with 10% FBS and 1% penicillin-streptomycin-Lglutamate (PSG). Cells were incubated in a 5% CO2 humidified
incubator at 37 °C and were typically seeded at 0.5 ⫻ 106/mL and
grown to a density of 2 ⫻ 106/mL.
Dimedone Labeling of Cell Lysate. Jurkat cells (6 ⫻ 106) were
collected by centrifugation (1,000 ⫻ g for 5 min) and washed
three times with sterile PBS. The media free cell pellet was then
suspended in 100 ␮L ice-cold lysis buffer (1.0% Nonidet P-40, 50
mM Tris-HCl, pH 8.0, 150 mM NaCl, and protease inhibitors)
and incubated on ice for 30 min with frequent mixing. The
supernatant was collected by centrifugation (20,000 ⫻ g for 20
min) at 4 °C. The supernatant was transferred to a new tube and
subjected to the same centrifugation protocol. The protein
concentration of the lysate was determined by Bradford assay
(Bio-Rad). Cell lysate (1 mg/mL, in 30-␮L total reaction volume)
was untreated or treated with 1, 2.5, 5, or 10 mM dimedone or
DMSO (2.5% wt/vol) for 1 h at room temperature. The reaction
was terminated by adding 10% ␤-mercaptoethanol in XT sample
buffer and analyzed by Western blot using the ␣-hapten antibody.
Comparative Labeling of GAPDH-SOH With Sulfenic Acid Probes.
described above. Cells were incubated in RPMI media containing 10 mM dimedone, 2% FBS, and 1% PSG at 37 °C for 0, 1,
2, 4, 6, and 8 h. At the end of each time point, cells (100 ␮L) were
mixed with trypan blue solution (100 ␮L) and quantified by
counting with a hemocytometer.
Dot Blot. PVDF membrane was treated with methanol for 10 min
before spotting proteins. Using narrow-mouth pipet tip, 1.3 ␮L
GADPH (0.31 mg/mL in PBS, pH 7.4) was spotted on the
membrane and dried for 5 min at room temperature. The
membrane was then untreated or incubated with 0.1 mM H2O2
for 5 min at room temperature and washed with PBS (2 ⫻ 10
min). Next, the membrane was incubated with 5 mM dimedone
in PBS or DMSO (2.5% vol/vol) for 15 min at room temperature.
The membrane was then washed with PBS (2 ⫻ 10 min), blocked
with 2% nonfat milk in TBST for 1 h at room temperature and
washed with TBST (3 ⫻ 10 min). The membrane was incubated
with ␣-hapten antibody in TBST (1:2,000) for 1 h at room
temperature, washed with TBST (3 ⫻ 10 min), and incubated
with goat ␣-rabbit-HRP (1:5,000) in TBST for 1 h at room
temperature. After washing with TBST (3 ⫻ 10 min), the
membrane was developed with ECL plus detection reagent.
Source and Preparation of Samples for Protein Microarray. Protein
Dimedone Labeling of Cells in Culture. Jurkat cells in RPMI with 2%
FBS and 1% PSG were incubated with 10 mM dimedone or
DMSO (5% vol/vol) for 0, 1, 2, 4, or 8 h at 37 °C. At the end of
each time point, cells were harvested, washed 3 times with PBS
and a lysate was generated and analyzed as described above.
Identification of Sulfenic Acid-Modified Proteins. Proteins were
immunoprecipitated from lysates made from dimedone-treated
cells using GAPDH, PrxI, and actin-specific antibodies (Santa
Cruz Biotechnology). After capture on protein A-Sepharose,
samples were analyzed by Western blot using the ␣-hapten
antibody.
Seo and Carroll www.pnas.org/cgi/content/short/0903015106
Biotechnologies Inc. provides pharmaceutical, biotechnology,
government, and academic institutions with human clinical
specimen derivatives and high-throughput protein and tissue
microarrays. The SomaPlex™ line of protein microarray research
products is prepared from normal and tumor tissue collected
from the same patient. Tissues for microarray research products
are obtained through a global network of participating medical
centers that employ IRB approved protocols and strict ethical
guidelines to ensure patient confidentiality and safety. Identical
procedures are used to prepare all patient samples. Specimens
are flash frozen to ⫺120 °C within 5 min of removal to minimize
autolysis, oxidation, and protein degradation. Tissue specimens
1 of ⴚ4
are homogenized in modified RIPA buffer (PBS, pH 7.4, 1 mM
EDTA, and protease inhibitors) to obtain the soluble proteins,
and centrifuged to clarify. Proteins are spotted in triplicate at a
protein concentration of 1 mg/mL and each spot contains
approximately 5 ng total protein. The proteins have not been
subjected to denaturing or reducing conditions. This allows the
microarray to be used in a variety of applications; to study
protein-protein interaction, ligand binding, and immunological
analysis for the detection of specific protein targets and posttranslational modifications (5–7).
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separations with moderate resolution. J Org Chem 43:2923–2925.
2. Khan AT, Ali MA, Goswami P, Choudhury LH (2006) A mild and regioselective method
for bromination of ␣-keto esters and 1,3-diketones using bromodimethylsulfonium
bromide (BDMS). J Org Chem 71:8961– 8963.
3. Dixon S, Dixon (2005) Solid-phase synthesis of quinoxaline, thiazine, and oxazine
analogs through a benzyne intermediate. Tetrahedron Lett 46:7443.
4. Seo YH, Carroll KS (2009) Facile synthesis and biological evaluation of a cell-permeable
probe to detect redox-regulated proteins. Bioorg Med Chem Lett 19:356 –359.
5. Hempel N, et al. (2007) Loss of betaglycan expression in ovarian cancer: Role in motility
and invasion. Cancer Res 67:5231–5238.
6. Gordon KJ, Dong M, Chislock EM, Fields TA, Blobe GC (2008) Loss of type III transforming growth factor beta receptor expression increases motility and invasiveness associated with epithelial to mesenchymal transition during pancreatic cancer progression.
Carcinogenesis 29:252–262.
7. Meyer R, et al. (2009) Overexpression and mislocalization of the chromosomal segregation protein separase in multiple human cancers. Clin Cancer Res 15:2703–2710.
8. Neve RM, et al. (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527.
Seo and Carroll www.pnas.org/cgi/content/short/0903015106
2 of ⴚ4
A
Dimedone
H2O2 (µM)
–
–
+
–
+
10
α-Hapten
Papain (Ponceau)
B
Dimedone
DAz-1-biotin
DAz-1
H2O2 (µM)
–
–
–
–
+
–
–
–
+
–
–
2.5
+
–
–
5.0
–
–
–
–
–
+
–
–
–
+
–
2.5
–
+
–
5.0
–
–
–
–
–
–
+
–
–
–
+
2.5
–
–
+
5.0
GAPDH–SOH
α-GAPDH
Fig. S1. Trapping and tagging sulfenic acid modified proteins. (A) Sulfenic acid-modified papain is detected by the ␣-hapten antibody. Top, papain (10 ␮M)
was treated with dimedone (0 or 5 mM) followed by H2O2 (0 or 10 ␮M) for 30 min at room temperature and analyzed by Western blot with the ␣-hapten antibody.
(B) Comparative labeling of sulfenic acid-modified proteins using dimedone, DAz-1-biotin and DAz-1. GAPDH (5 ␮M in PBS, pH 7.4) was treated with 500 ␮M
dimedone, 500 ␮M DAz-1-biotin, 500 ␮M DAz-1, or DMSO (2.5% vol/vol) followed by H2O2 (0, 2.5, or 5 ␮M) for 30 min at room temperature. DAz-1 treated GAPDH
was further incubated with 250 ␮M p-biotin for 2 h at 37 °C. Proteins were resolved by SDS/PAGE using Criterion XT 4⫺12% Bis-Tris gels (Bio-Rad) in XT Mes
running buffer, transferred to PVDF membrane, and then blocked with 5% nonfat dried milk in PBST overnight at 4 °C or for 1 h at room temperature. The
membrane was then washed in PBST (2 ⫻ 10 min) and probed with ␣-hapten antibody (1:200) or HRP-streptavidin (1:100,000) for 1 h at room temperature. The
membrane was then washed with PBST (3 ⫻ 10 min) and incubated with goat ␣-rabbit-HRP (1:100,000) for 1 h at room temperature, washed with PBST (3 ⫻ 10
min), and developed using ECL Plus detection reagent (GE Healthcare). Equal protein loading was verified by probing with ␣-GAPDH.
Seo and Carroll www.pnas.org/cgi/content/short/0903015106
3 of ⴚ4
A
B
C
Time
Dimedone
50
250
250
100
100
Dead Cells (%)
40
30
50
50
37
Actin
GAPDH
37
Prx
20
Actin
GAPDH
*
20
20
15
10
*
α-GAPDH
Prx
15
Ponceau
0
0
1
2
4
6
8
Time (hr)
Fig. S2. ␣-Hapten antibody specifically recognizes sulfenic acid-modified proteins in vitro and in vivo. (A) Viability assay of Jurkat cells treated with dimedone.
Jurkat cells were incubated in media containing 10 mM dimedone for 0, 1, 2, 4, 6, and 8 h. At the end of each time point, cells (100 ␮L) were mixed with trypan
blue solution (100 ␮L) and dead cells were quantified by counting with a hemocytometer. (B) Dose-dependence of dimedone labeling. Jurkat cell lysate (1 mg/mL)
was treated with dimedone (0, 1, 2.5, 5, or 10 mM) for 1 h at room temperature and analyzed by Western blot with the ␣-hapten antibody. Bands corresponding
to actin, GAPDH, and PrxI are indicated on the right. (C) Time-dependence of dimedone labeling. Jurkat cells were incubated in media containing 5 mM dimedone
for 0, 1, 2, 4, and 8 h. Cell lysate was analyzed by Western blot with the ␣-hapten antibody. Bands corresponding to actin, GAPDH and PrxI are indicated on the
right. Asterisks denote a background immunoreactive band.
Seo and Carroll www.pnas.org/cgi/content/short/0903015106
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Dimedone
H2O2 (µM)
–
–
–
100
+
–
+
100
α-Hapten
GAPDH (Ponceau)
Fig. S3. Detecting thiol oxidation in GAPDH in dot blot format. Sulfenic acid-modified GAPDH is detectable with the dot blot format. Top, PVDF membrane
as spotted with GAPDH (0.4 ␮g) was untreated or incubated with 0.1 mM H2O2 followed by 5 mM dimedone of DMSO (2.5% vol/vol). The dot blot was then
analyzed with the ␣-hapten antibody.
Seo and Carroll www.pnas.org/cgi/content/short/0903015106
5 of ⴚ4
A
B
2 107
2 10
*
*
7
**
Fluorescence (a.u.)
**
5 10
6
*
*
0
1 107
5 106
Patient 2
Patient 3
ed
Tu
m
or
at
M
Patient 1
ch
ed
Tu
m
or
M
at
ch
or
ed
Tu
m
ch
M
at
M
at
ch
ed
Tu
m
or
0
Patient 4
Transitional cell carcinoma
ch
e
Tu d
m
or
M
at
ch
e
Tu d
m
or
M
at
ch
e
Tu d
m
or
M
at
ch
e
Tu d
m
or
M
at
ch
e
Tu d
m
or
7
at
1 10
M
Fluorescence (a.u.)
1.5 107
*
*
1.5 107
Patient 5
Patient 6
Squamous cell
carcinoma
Patient 7
Patient 8
Patient 9
Epidermoid Adenocarcinoma carcinoma
Fig. S4. Comparison of sulfenic acid levels in bladder tumors to corresponding matched normal tissue. The protein microarray was incubated in buffer
containing 2 mM dimedone for 1 h and probed for sulfenic acids with the ␣-hapten antibody. The fluorescence intensities of microarray spots are indicated with
bars. Error bars represent s.d. of three replicate spots. (A) Transitional cell carcinoma tissue and matched normal tissue in four patient samples. (B) Squamous
cell, epidermoid, and adenocarcinoma and matched normal tissue in patient samples. The Student’s t test was used to determine statistical significance of the
observed differences (*, P ⬍ 0.001; **, P ⬍ 0.005).
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O
Br
O
O
NaHCO 3
O
+
piperidine
NHFmoc
HS
3
70%
O
5
S
+
OH
O
F
F
pyridine
O
F
CH 2 Cl2 , rt
30 min, Ar
81 %
O
O
S
F3 C
F
7
6
HS
1
Fig. S5.
O
O
8
DIEA, DMF
50 o C, 4 h, Ar
88%
H2 N
F
F
S
5
O
O
S
S
O
A
O
(1) 1 N NaOH
N
H
F
O
O
O
O
F
F
O
S
DMF, 35%
4
2
H2 N
S
FmocHN
THF, rt, 2 h
(2) DTT
68 % in two steps
O
N
H
S
O
9
Chemical synthesis of the hapten conjugate used to elicit sulfenic acid-specific antibodies.
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Table S1. Source, clinical, and pathological features of tumors used to derive breast cancer cell lines used in this study, adapted from
ref. 8
Cell line
1
2
3
4
5
6
BT20
BT474
HS578T
MCF7
MDAMB231
MDAMB468
Gene cluster
BaA
Lu
BaB
Lu
BaB
BaA
ER
⫺
⫹
⫺
⫹
⫺
⫺
PR
[⫺]
[⫹]
[⫺]
[⫹]
[⫺]
[⫺]
HER2
⫹
TP53
Source
Tumor type
Age
Ethnicity
Culture media
Culture condition
⫹⫹WT
P.Br
P.Br
P.Br
PE
PE
PE
IDC
IDC
IDC
IDC
AC
AC
74
60
74
69
51
51
W
W
W
W
W
B
DMEM, 10% FBS
RPMI, 10% FBS
DMEM, 10% FBS
DMEM, 10% FBS
L15, 10% FBS
L15, 10% FBS
37 °C, 5% CO2
37 °C, 5% CO2
37 °C, 5% CO2
37 °C, 5% CO2
37 °C, no CO2
37 °C, no CO2
⫹
⫹M
⫹/⫺WT
⫹⫹M
[⫹]
AC, adenocarcinoma; BaA, Basal A; BaB, Basal B; IDC, invasive ductal carcinoma; Lu, Luminal; P.Br, primary breast; PE, pleural effusion; W, White; B, Black.
ER/PR/HER2/TP53 status: ER/PR positivity, HER2 overexpression, and TP53 protein levels and mutational status (M, mutant protein; WT, wild-type protein) are
indicated. Square brackets indicate that levels are inferred from mRNA levels alone where protein data is not available. Media conditions: FBS, fetal bovine serum;
DMEM, Dulbecco’s modified Eagle’s medium, GIBCO #11965– 092; RPMI, RPMI medium 1640, GIBCO #27016 – 021; L15, Leibovitz’s L-15 medium, GIBCO
#11415– 064.
Other Supporting Information Files
SI Appendix
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