Quantitative Profiling of Protein Carbonylations in Ferroptosis by an Aniline-Derived Probe

Article abstract of DOI:10.1021/jacs.8b01462

Ferroptosis is a regulated form of necrotic cell death implicated in carcinogenesis and neurodegeneration that is driven by phospholipid peroxidation. Lipid-derived electrophiles (LDEs) generated during this process can covalently modify proteins ("carbonylation") and affect their functions. Here we report the development of a quantitative chemoproteomic method to profile carbonylations in ferroptosis by an aniline-derived probe. Using the method, we established a global portrait of protein carbonylations in ferroptosis with >400 endogenously modified proteins and for the first time, identified >20 residue sites with endogenous LDE modifications in ferroptotic cells. Specifically, we discovered and validated a novel cysteine site of modification on voltage-dependent anion-selective channel protein 2 (VDAC2) that might play an important role in sensitizing LDE signals and mediating ferroptosis. Our results will contribute to the understanding of ferroptotic signaling and pathogenesis and provide potential biomarkers for ferroptosis detection.

Full text of DOI:10.1021/jacs.8b01462

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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Quantitative Profiling of Protein Carbonylations in Ferroptosis by an
Aniline-Derived Probe
Ying Chen,†,‡ Yuan Liu,†,‡,§ Tong Lan,†,‡ Wei Qin,†,§ Yuntao Zhu,†,‡ Ke Qin,†,‡ Jinjun Gao,†,§
Haobo Wang,†,‡ Xiaomeng Hou,†,‡ Nan Chen,†,‡ Jose Pedro Friedmann Angeli,∥,⊥ Marcus Conrad,∥
and Chu Wang*,†,‡,§
†Synthetic and Functional Biomolecules Center, Beijing National ‡Laboratory for Molecular Sciences, Key Laboratory of Bioorganic
Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, and
§Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
∥Institute of Developmental Genetics, Helmholtz Zentrum Munchen, Munchen, Germany
S
* Supporting Information
ABSTRACT: Ferroptosis is a regulated form of necrotic cell
death implicated in carcinogenesis and neurodegeneration that
is driven by phospholipid peroxidation. Lipid-derived electro-
philes (LDEs) generated during this process can covalently
modify proteins (“carbonylation”) and affect their functions.
Here we report the development of a quantitative chemo-
proteomic method to profile carbonylations in ferroptosis by
an aniline-derived probe. Using the method, we established a
global portrait of protein carbonylations in ferroptosis with
>400 endogenously modified proteins and for the first time, identified >20 residue sites with endogenous LDE modifications in
ferroptotic cells. Specifically, we discovered and validated a novel cysteine site of modification on voltage-dependent anion-
selective channel protein 2 (VDAC2) that might play an important role in sensitizing LDE signals and mediating ferroptosis. Our
results will contribute to the understanding of ferroptotic signaling and pathogenesis and provide potential biomarkers for
ferroptosis detection.
addition of a nucleophilic amino acid (cysteine, histidine, or
lysine) by an electrophilic α,β-unsaturated aldehyde produced
during lipid peroxidation,8−11 leaving a reactive “carbonyl”
group on the target protein. Recent studies have shown that the
intracellular concentration of malondialdehyde (MDA), a bona
fide lipid-derived electrophile (LDE),12 is increased when cells
succumb to ferroptosis,1,13 which could potentially result in
elevated protein carbonylation and augment ferroptotic signal-
ing. However, the protein targets and specific sites of
carbonylation and their roles in ferroptosis remain unexplored.
Several methods have been developed to detect protein
carbonylation.14−16 Cravatt and colleagues reported a com-
petitive activity-based protein profiling (ABPP17) strategy to
profile targets of LDE modification in cell lysates using a
cysteine-reactive iodoacetamide-alkyne (IAyne) probe.18
Although the method is able to quantify >1000 LDE-sensitive
cysteines in proteomes, it suffers from the indirect detection of
modifications on only cysteines but not other nucleophilic
residues. Bioorthogonal LDE-analogue or precursor probes
such as HNE-alkyne have been synthesized and applied to label
and enrich targets of protein carbonylation.19−22 However,
these unnatural surrogate probes are not suitable for detecting
INTRODUCTION
■
Ferroptosis is a newly annotated form of cell death that has
been implicated with diseases such as acute kidney injury,
Huntington’s disease, cancer, pericentricular leukomalacia, and
SECIS-binding protein 2 (SBP2) deficiency syndrome.1,2
Ferroptosis is morphologically, biochemically, and genetically
distinct from other forms of cell death, such as apoptosis,
necroptosis, and autophagy.3−5 Classical hallmarks of ferropto-
sis include the dependence on intracellular iron and
accumulation of phospholipid hydroperoxides.1,5−7 Two
small-molecule ligands have been reported to induce ferroptosis
with validated mechanisms. One is erastin which inhibits the
cell surface cystine-glutamate antiporter (system xc−) resulting
in depletion of reduced glutathione and subsequent inactivation
of glutathione peroxidase 4 (GPX4).3 The other is RSL3 that
can induce ferroptosis without impacting on GSH level by
covalently modifying and inhibiting the activity of GPX4. Due
to the unique function of GPX4 in reducing phospholipid
hydroperoxides, both erastin and RSL3 are able to trigger the
process of lipid peroxidation that culminates with cell death.5,6
It has been well-known that the process of phospholipid
oxidation is able to generate diffusible electrophilic species able
to induce specific modifications on proteins that can perturb
their activity and function. One major type is often dubbed as
“protein carbonylation”, which could be derived by Michael
Received: February 8, 2018
© XXXX American Chemical Society
A
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Figure 1. Evaluating the reactivity of aniline probes to detect protein carbonylation in native proteomes. (a) A general scheme of capturing HNE-
carbonylated proteins by aniline probes. (b) Comparison of the reactivity of o-, m-, p-APA probes with proteome carbonylations induced by HNE.
(c) The reduction by NaBH3CN is critical in m-APA labeling. (d) Detection of concentration-dependent HNE-carbonylations in proteomes by m-
APA. (e) The labeling of m-APA can be effectively competed by free aniline. (f) Imaging of protein carbonylations in HNE-treated cells using m-APA
by fluorescence confocal microscopy. (g) Confirmation of the adduct of m-APA with HNE carbonylated glutathione by LC-MS. Shown are the
extracted ion chromatographic traces of GSH, the GSH-HNE adduct, and the final product formed with m-APA.
endogenous modification events in cells. Due to its chemical
nature, protein carbonylation formed through Michael addition
will leave a reactive aldehyde or ketone group on protein targets
which enables the selective capture of these modifications with
carbonyl-directed ligation probes.
Herein, we report the development of a unique chemo-
proteomic platform to profile protein carbonylations using an
aniline-based probe. We first validated the approach in the cell
lysates modified with 4-hydroxy-2-nonenal (HNE), a well-
studied LDE molecule that can cause protein carbonylation,
and then applied our method in detecting endogenous target
proteins and residue sites of protein carbonylation during
ferroptosis.
Hydrazide23−26 and aminooxy probes27−30 are the most
popular choices for capturing protein carbonylations in
proteomes. These methods are based on chemoselective
imine ligations of aminooxy or hydrazide with aldehyde/ketone
to form the covalent oxime or hydrazone adduct, which are
widely used in drug delivery, protein labeling and PEGyla-
tion.31−39 Imine formation originally suffered from slow
reaction rates which often requires reaction with high
concentration of reactants and at acidic conditions, a critical
challenge for many biological applications.32 The limitation was
lifted by Dawson and co-workers by their revolutionary
discovery of aniline as catalyst to significantly accelerate
oxime ligation and hydrazone formation.40−42 Enhancement
of reaction rates follows the mechanism of nucleophilic catalysis
where a highly reactive intermediate of protonated aniline
Schiff base was quickly formed with aldehyde substrates and a
“transimination” reaction then occurs to form the final
hydrazone/oxime product. Notably, the aniline Schiff base
intermediate can be stabilized by reduction of the imine bond
with NaBH3CN, forming the aniline-modified peptide sub-
strates,41 and this chemistry has also been used in derivatization
of aldehyde-containing metabolites and drug intermedi-
ates.43−50 Inspired by these observations, we reasoned that
aniline, due to its rapid reaction kinetics and high chemo-
selectivity with aldehydes, should be applicable as a warhead to
react directly with protein targets of carbonylation (Figure 1a).
RESULTS AND DISCUSSION
■
Evaluating the Reactivity of Aniline Probes to Detect
Protein Carbonylation in Native Proteomes. Conven-
iently, commercially available aminophenylacetylene (APA)
compounds with the alkyne group at ortho, para, and meta
positions, respectively (Figure 1a), can be readily tested for
their efficiency to capture protein carbonylations from cell
lysates. Proteomes from nonsmall cell lung cancer H1299 cells
were treated with 100 μM of HNE and then labeled with 0.5
mM of each of the APA probes. After reduction with
NaBH3CN, the APA-labeled proteins were conjugated with
fluorophore-N3 via copper-catalyzed click chemistry51 and
visualized by in-gel fluorescence. Among the three APA probes,
m-APA exhibited the best efficiency in reacting with HNE-
modified proteomes (Figure 1b). This can be explained by the
decreasing electron-withdrawing ability when the alkyne is at
the meta position, which gives the strongest nucleophilicity on
the aniline warhead. We further found that the optimal pH for
m-APA labeling is between 5 and 6 (Figure S1a of the
Supporting Information, SI), which is consistent with the fact
that acidic conditions favor the formation of imine. It is also
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Figure 2. Quantitative profiling of HNE-sensitive sites in proteomes using m-APA by isoTOP-ABPP. (a) Scheme of quantifying the sensitivity of
HNE modification in proteomes by isoTOP-ABPP. The extent of HNE modifications upon low and high concentrations of HNE treatment was
quantified for each site and more saturated labeling (ratio ≈ 1) indicates higher sensitivity toward HNE modification. (b) Distribution of the
isoTOP-ABPP ratios for ∼1000 cysteines in proteomes upon treatment with 20 and 100 μM HNE. The inlet shows the zoom-in view of the group of
HNE-hyper-sensitive cysteines (ratio ≤2.0, red boxed) with representative sites marked (C22 of ZAK, C1001 of RTN4, C282 of HMOX2, and C210
of VDAC2). (c) Gene ontology analysis of proteins harboring HNE-hyper-sensitive cysteines in terms of their molecular functions. (d) MS/MS
spectra supporting the HNE modification on C282 of HMOX2 and C210 of VDAC2. (e) m-APA selectively labeled carbonylations on C210 of
VDAC2 and C282 of HMOX2 in proteomes when cells are treated with increasing concentrations of HNE.
1
critical to stabilize the imine bond with the reduction step by
NaBH3CN (Figure 1c). The labeling reached saturation at 0.5
mM of m-APA and was completed within 60 min (Figures S1b,
S2a, and S2b). With these optimized conditions, we showed
that m-APA was able to monitor protein carbonylations in
proteomes induced with various concentrations of HNE
(Figure 1d). The labeling could be competed by free aniline
(Figure 1e) and was not contaminated by other oxidative forms
of cysteine modification, such as protein sulfenylation (Figure
S1c). We also treated cells in situ with HNE and demonstrated
that m-APA can efficiently label carbonylated proteins in fixed
cells, enabling direct imaging using confocal microscopy
(Figure 1f). In addition, we used the tripeptide glutathione
(GSH) as a model to validate the reaction between m-APA and
carbonylated cysteines. GSH was reacted with 100 μM HNE to
form a Michael adduct bearing a free aldehyde and 0.5 mM of
m-APA was added to continue reaction for 60 min. After
reduction of the imine bond with 60 mM NaBH3CN, we
successfully detected the GSH-HNE-m-APA product (m/z =
563) by UPLC-ESI-MS (Figure 1g). The product was further
purified by HPLC and characterized by Fourier Transform Ion
Cyclotron Resonance Mass Spectrometry (FTMS) with a
correct mass (Figure S26). Lastly, we validated this reaction
using a small-molecule substrate, N-acetyl-L-cysteine (NAC),
and characterized the final adduct by H NMR, 13C NMR as
well as HRMS (Figure S27, S28, S29, and S30), all of which
confirm that m-APA is able to capture carbonylated cysteines as
predicted.
Identifying Sites of Carbonylation Using a Tandem
Orthogonal Proteolysis (TOP)-ABPP Strategy.52 We
continued to combine m-APA labeling and TOP-ABPP strategy
for identifying HNE modified sites. After proteomes were
treated with 100 μM HNE and labeled by m-APA, an azide-
biotin tag bearing a cleavable azobenzene site (“azo-biotin”)53
was conjugated with the m-APA labeled proteins. Following the
streptavidin enrichment and on-bead trypsin digestion, an
orthogonal round of cleavage was performed by sodium
dithionite to release the HNE-modified peptides from the
streptavidin beads, which were then analyzed by LC-MS/MS
(Figure S3a). We performed three biological replicates and
collectively identified >1000 cysteines, 25 histidines and 5
lysines as sites of HNE modifications, which is consistent with
previous studies that cysteines are major sites of modifications
by HNE through Michael addition19,54,55 (Figure S3c, Table
S1). We also compared the performance of m-APA with two
other previously reported clickable probes, HZyne and
AOyne,27 side by side. HZyne did not identify any site under
the same experimental condition, which is expected from its
C
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Figure 3. Quantifying endogenous protein carbonylations in ferroptosis using m-APA with RD-ABPP. (a) Lipid peroxidation plays an important role
in mediating ferroptosis as RSL3 can induce ferroptosis by inhibiting GPX4, a critical detoxifying enzyme for lipid peroxidation and Lip-1 can rescue
cells from ferroptosis by scavenging products from lipid peroxidation. (b) Detection of protein carbonylations in ferroptotic HT1080 cells after
induced with 0, 50, 150 nM RSL3 and/or rescued by 300 nM Lip-1. (c) A venn diagram showing the number of carbonylated proteins identified in
ferroptotic cells vs control or rescued cells. (d) Disease analysis of carbonylated proteins in ferroptosis. (e) Cellular component analysis of
carbonylated proteins in ferroptosis. (f) Raw chromatographic peaks of selected proteins quantified with carbonylation in ferroptosis. Red and blue
are traces of light and heavy dimethylated peptides with protein names and quantified ratios shown above and below, respectively.
weakest labeling as evaluated by in-gel fluorescence (Figure
S2a, b). Surprisingly, despite its seemingly better labeling
efficiency, AOyne identified only 293 cysteines, 15 histidines
and 4 lysines as sites of HNE modifications by TOP-ABPP,
which are much less than those identified by m-APA (Figure
S2a, b and S3b). Further investigation showed that the AOyne-
modified peptide adducts were unstable, which were broken at
the N−O bond during sample preparation, probably due to a
combined condition of high temperature and acidic buffer
(Figure S4). These data suggested that m-APA is a superior
probe for the purpose of labeling protein carbonylations for
chemical proteomics analysis by LC-MS/MS. Given cysteines
were found as the major sites of HNE modifications, we
performed secondary structure feature analysis on these HNE-
adducted peptides and the results showed a marked preference
of HNE for cysteines at coil instead of α-helix and β-sheet
(Figure S5a). Interestingly, alignment of local sequences
flanking the site of modifications revealed that HNE prefers
to modify cysteines when a second cysteine is not available at
position X+2 or X−2 (Figure S5b), which is the representative
motif (CXXC) of the thioredoxin family. This result suggests
that despite their heightened intrinsic reactivity, proteins with a
thioredoxin like fold are not preferred targets for HNE
modification, which are consistent with our previous finding
using the IAyne probe.18 Among the cysteines identified are
several previously reported sites of HNE modification,18
including C1101 of reticulon-4 (RTN4) and C152 of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure
S6b). We overexpressed these proteins along with their cysteine
mutants in HEK-293T cells and immunoblotting showed that,
upon HNE treatment, the m-APA probe could only capture the
wild-type proteins, but not any of the cysteine mutants (Figure
S6a, c). These results not only biochemically confirmed these
cysteines as the specific sites for HNE modification, but also
highlighted the efficiency of the m-APA probe in selectively
labeling protein carbonylations in complex proteomes.
Quantitatively Ranking the HNE Sensitivity by an
Isotopic TOP-ABPP Method (“isoTOP-ABPP”).56 In order
to quantitatively rank HNE-sensitive cysteines in proteomes,
we next applied the m-APA labeling to compare the levels of
modification at each site upon treatment with variable
concentrations of HNE, a strategy which has been used to
profile intrinsic chemical reactivity of cysteines in proteomes.56
Proteomes from H1299 cells were treated with low (20 μM)
and high (100 μM) concentrations of HNE independently,
labeled with the m-APA and conjugated with isotopically
labeled heavy and light azo-biotin tags, respectively. The
proteomes were combined, enriched, digested by trypsin and
cleaved by sodium dithionite before LC-MS/MS analysis. The
quantitative ratio (light/heavy) “R” for each HNE-adducted
peptide reflects its sensitivity toward HNE modification with
lower ratio corresponding to higher HNE sensitivity (i.e., the
HNE labeling gets closer to saturation upon treatment with low
concentration of HNE) (Figure 2a). We quantified ∼1000
D
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Table 1. Summary of Protein Targets Which Have Potential Links with Ferroptosis in RD-ABPP Results
uniprot #
gene name
ratio (RLS/DMSO)
ratio (+Lip-1/−Lip-1)
potential link with ferroptosis
ref
P02786
Q658P3
P45880
Q9Y277
O60488
P01116
TFRC
3.18
2.44
2.71
2.09
10.71
7.75
2.9
transferrin receptor, with increased expression in ferrotosis-sensitive cells
function of reducing Fe3+ to ferrous iron
57
2
STEAP3
VDAC2
VDAC3
ACSL4
KRAS
12.57
10.62
3.12
positive regulator of ferroptosis, direct target of erastin
69
69
67
68
positive regulator of ferroptosis, direct target of erastin
regulation of ferroptosis sensitivity by shaping cellular lipid composition
oncogenic RAS mutant cells sensitive to ferroptosis induced by erastin
11.82
7.56
cysteines with various isoTOP-ABPP ratios and using R ≤ 2 as
the cutoff, we were able to define ∼100 hyper-sensitive
cysteines toward HNE modification in proteomes (Figure 2b,
Table S2). Analysis of the proteins harboring these HNE-
hyper-sensitive cysteines revealed that porin activity is the most
significantly enriched molecular function, followed by Ran
GTPase binding activity and peptide/protein transporter
activity (Figure 2c). We compared the HNE-hyper-sensitive
cysteines in our list with those obtained previously using a
competitive ABPP method and found that 4 of the top 5 HNE-
sensitive cysteines in that list were also identified by our current
approach (Figure 2b, Table S3), including C24 of FN3KRP,
C22 of ZAK, C1001 of RTN4 and C41 of EEF2.
In addition, we also found several novel HNE-hyper-sensitive
cysteines including C210 of voltage-dependent anion-selective
channel protein 2 (VDAC2) and C282 of heme oxygenase 2
(HMOX2) (Figure 2d). We overexpressed these two newly
identified proteins along with their cysteine mutants in HEK-
293T cells and the concentration-dependent HNE treatment of
living cells showed that the hyper-sensitive cysteines could be
detected at as low as 5 μM of HNE (Figure 2e). Collectively,
these results, using HNE as a model compound, demonstrated
that m-APA is a powerful and efficient probe to capture sites of
protein carbonylation both in vitro and in situ for chemical
proteomic profiling.
Profiling of the Endogenous Events of Protein
Carbonylations in Ferroptosis. Ferroptosis is a newly
defined type of cell death that is hallmarked by its dependence
on intracellular iron and exacerbated lipid peroxidation. RSL3, a
potent inhibitor of GPX4, which is responsible for reducing
lipid hydroperoxides to the corresponding alcohols, can
potently trigger ferroptosis in HT1080 cells.57 The cell death
can also be efficiently suppressed by liproxstatin-1 (Lip-1), a
spiroquinoxalinamine derivative that functions as a catalytic
ROS scavenger7,58,59 (Figure 3a). Consistent with these reports,
when we treated HT1080 cells with RSL3 to induce ferroptosis,
we observed an increase in lipid ROS which could be blocked
by pretreatment of Lip-1 (Figure S7a). We therefore applied
the m-APA probe in the ferroptotic condition aiming to capture
protein carbonylations during this process. Lysates from RSL3-
treated and/or Lip-1-treated HT1080 cells were labeled with m-
APA and conjugated with fluorophore-azide for analysis by in-
gel fluorescence. The fluorescence signals increased with the
increasing concentrations of RSL3, which supports elevated
lipid peroxidation and protein carbonylation in ferroptosis.
When Lip-1 was added to rescue cells from ferroptosis, we also
observed a concomitant decrease in the m-APA labeling signal
(Figure 3b), corroborating that the m-APA signal detected was
indeed derived from endogenously produced LDEs.
ABPP experiments were designed and performed (Figure S7b).
In one experiment, proteomes from control cells and
ferroptotic cells were labeled by the m-APA probe and the
resulting digested peptide samples were subjected to reductive
dimethylations by heavy or light formaldehydes, respectively. In
the other experiment, proteomes from RSL3-treated cells with
and without the ferroptosis inhibitor Lip-1 were labeled with m-
APA instead. We defined proteins that were quantified in at
least two replicates with a light/heavy ratio ≥2 as potential
targets and collectively identified 1075 and 1029 carbonylated
proteins in each type of the experiments, respectively, with a
total number of 461 proteins in common (Figure 3c and Table
S4). This data set constitutes, to the best of our knowledge, the
first global inventory of carbonylated protein targets modified
by endogenous products of lipid peroxidation in ferroptosis.
Gene Ontology (GO) Analysis of Carbonylated
Targets in Ferroptosis. GO analysis of the data set revealed
that the carbonylated proteins are dominantly localized in the
membrane compartments, including Endoplasmic Reticulum
(ER) membranes, Golgi apparatus as well as mitochondrial
membranes (Figure 3e), which are highly enriched with poly
unsaturated fatty acids (PUFAs) that are prone to lipid
peroxidation.62 Interestingly, ER has been shown as a major site
of lipid peroxidation during ferroptosis,7 and it is anticipated
that the lipid electrophiles might preferentially target proteins
by proximity effect. We also analyzed these carbonylated
proteins based on annotations recorded in the DAVID disease
database and found that they are highly enriched in diseases
such as lung cancer, kidney failure, and Alzheimer’s disease
(Figure 3d). Most of these diseases have been previously
implicated with ferroptosis1,2,58,63,64 and given that HIV
infection can cause dysregulation of glutathione homeo-
stasis65,66 and induce lipid peroxidation, it is not surprising to
find AIDS/HIV infections on the top of the list, too.
Among the list of carbonylated proteins, we found several
known targets that have potential links with ferroptosis (Figure
3f, Table 1). For example, ACSL4 has been shown to dictate
ferroptosis sensitivity by shaping cellular lipid composition.67
The oncogenic mutant cell lines of KRAS can be selectively
induced into ferroptosis by RSL3.68 VDAC2/3 were previously
identified as the direct targets of erastin, another potent
ferroptosis-inducing compound.69 Notably, our data set also
revealed that a couple of vital proteins involved in iron
metabolism are carbonylated during ferroptosis including
TFRC,
a transferrin receptor, and STEAP3, a
ferrireductase.2,57 Normally, Fe3+ is imported into cells through
TFRC and is then located in the endosome where it is reduced
to Fe2+ by STEAP3. So it is possible that carbonylation of
TFRC and STEAP3 might impede their functions to slow
down the overload of Fe2+ and reduce its role in catalyzing the
ferroptosis process via Fenton reaction. This may act as a
negative feedback loop to defend cells from excessive lipid
peroxidation as much as possible.
In order to quantitatively assess targets of carbonylation in
ferroptosis, we implemented a quantitative chemical proteomic
platform named “RD-ABPP” that combines reductive dime-
thylation (“RD”) coupled to ABPP.60,61 Two independent RD-
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Figure 4. Identification and validation of endogenous LDE modifications in ferroptosis. (a) Scheme of identifying endogenous LDE modified sites in
ferroptosis using TOP-ABPP strategy. m-APA labeled proteomes were conjugated with an acid cleavable azide-biotin tag and the LDE modified
peptides were released by incubation with 2% formic acid for LC-MS/MS analysis. (b) List of selected residue sites with endogenous HNE/ONE
modifications in ferroptosis. The full list can be found in Table S5. (c) Validating the endogenous carbonylation on C210 of VDAC2 during
ferroptosis as the m-APA labeling was lost upon mutation of C210 to alanine. Immunoblotting (top) and signal quantification (bottom) of 6xHis-
tagged wildtype VDAC2 and its C210A mutant before (“input”) and after (“output”) m-APA labeling and enrichment. (d) Cell viability test of three
HT1080 stable cell lines overexpressing GFP, wildtype VDAC2 and its C210A mutant in the course of ferroptosis induced by RSL3. Values
represent means SD from six replicate experiments. * p < 0.05, ** p < 0.01, Student’s t test.
Identification and Validation of Endogenous Sites of
LDE Modifications in Ferroptosis. In addition to identifying
carbonylated proteins by products of lipid peroxidation in
ferroptosis, it will be highly desired to locate the specific sites
with endogenous LDE modifications. Technically, this is a
more challenging task because such endogenous modifications
are often substoichiometric and unlike protein identification
with multiple tryptic peptides, site-specific mapping can only
rely on one adducted peptide. After our initial attempt using
the azo-benzene enrichment tag failed to identify any
endogenously modified sites (data not shown), we turned to
an alternative acid cleavable azide-biotin tag which was
previously shown to be more robust and efficient to recover
probe-labeled peptides in chemoproteomic analysis of protein
glycosylations70−72 (Figure 4a). In the new experimental
setting, the peptides were released from the beads through
incubation with 2% formic acid and analyzed by LC-MS/MS.
We searched the resulting LC-MS/MS data using masses of
common LDE adducts (HNE, ACR, ONE, HHE, OHE, and
MDA) with multiple filters (see methods), and finally identified
8 endogenous sites of modification by HNE, 11 sites by ACR,
and 5 sites by ONE (Figure 4b). This represents, to our best
knowledge, the first list of sites of modification by endogenous
LDEs in ferroptosis, which will be highly valuable for
manifesting the links between lipid peroxidation and
ferroptosis.
Among the list of sites of endogenous LDE modifications,
VDAC2 attracted our attention because its C210 was identified
as hyper-sensitive site toward exogenous HNE modification
and the protein was highly enriched by the m-APA probe when
cells were induced with ferroptosis. More strikingly, C210 was
endogenously modified by both HNE and ONE during
ferroptosis as unambiguously evidenced by MS/MS spectra
(Figure S8). We therefore set out to biochemically validate and
functionally characterized the carbonylations on C210 of
VDAC2 during cell ferroptosis. We constructed HT1080 cell
lines with stable overexpression of wildtype VDAC2 or its
C210A mutant. Upon ferroptosis induced by RSL3, the m-APA
probe could only label the wildtype VDAC2 but not the C210A
mutant, confirming carbonylations existing on this specific site
(Figure 4c). We next compared the viability of three HT1080
cell lines with stable overexpression of GFP, wildtype VDAC2
and its C210A mutant in the course of ferroptosis induced by
RSL3. We found that overexpression of VDAC2WT but not
VDAC2C210A could partially protect cells from ferroptosis
(Figure 4d), possibly by quenching the LDE signals during this
process. These data suggested that endogenous LDE
modifications on C210 of VDAC2 are functionally implicated
with sensitizing cells to ferroptosis upon RSL3-induced
oxidative stress. More thorough biological characterizations
remain to be performed to unveil the mechanistic insights
linking between these events of LDE modifications and
induction of ferroptosis.
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LSM Confocal Microscopy. HT1080 cells were grown to 70%
confluence and incubated with 20 μM HNE for 1 h with serum-free
medium. The cells were washed with prewarmed PBS, fixed with PBS
containing 4% (v/v) formaldehyde at 37 °C for 15 min, rinsed three
times with PBS and permeabilized in PBS containing 0.2% (v/v)
TritonX-100 at 37 °C for 10 min. The cells were again rinsed three
times with PBS and labeled with 0.5 mM m-APA for 1 h. Afterward,
the cells were washed three times using PBS with gentle shaking, and
clicked with 100 μM BTTAA-CuSO4 complex, 100 μM fluorophore-
N3 and 2.5 mM sodium ascorbate for 15 min at room temperature.
Finally, the cells were stained with 25 μg/mL Hoechst 33342 for 10
min to indicate the nucleus region. Images were acquired with Zeiss
LSM 700 laser scanning confocal microscope equipped with 63×/1.4
NA oil immersion objective lens.
CONCLUSIONS
■
In summary, we have reported the development and application
of an aniline-based probe, m-APA, to chemoselectively capture
protein carbonylations in proteomes. Using m-APA, we directly
identified in native proteomes >1000 sites of modification by
HNE, a bona fide LDE, and quantitatively ranked their
reactivity. We further applied this chemoproteomic strategy
to generate, to our best knowledge, the first global portrait of
protein carbonylations in the course of ferroptosis with >400
endogenously LDE-modified proteins and >20 modified sites.
We also biochemically validated a functional cysteine C210 in
the mitochondria outer membrane (OMM) anchored channel
protein VDAC2 as an endogenous site of LDE modifications
which might play an important role in sensitizing LDE signals
and mediating cells to ferroptosis. These efforts will facilitate
further investigation of the molecular mechanism of ferroptosis
and provide a valuable path for biomarker discovery in
ferroptosis. Given the high labeling efficiency and chemo-
selectivity of the m-APA probe which is readily available from
commercial sources with little cost, we envision that it will be
an ideal tool for detecting and capturing protein carbonylations
in many other physiological and/or pathological conditions,
where ferroptosis, oxidative stress, and lipid peroxidation are
centrally involved including neurodegeneration and inflamma-
tion.
Identification and Quantification of Exogenously HNE
Modified Sites. For identification of HNE modified sites, proteomes
treated with 100 μM HNE were labeled by 0.5 mM m-APA for 60 min
(labeling condition of HZyne and AOyne was same as m-APA, except
that NaBH3CN was not needed in the case of AOyne labeling). The
proteomes were then reacted with 1 mM CuSO4, 100 μM TBTA
ligand, 100 μM biotin-AZO-N3 (light) tag, and 1 mM TCEP for 1 h.
The proteomes were precipitated with methanol/chloroform,
resuspended in 1.2% SDS/PBS, and diluted to 0.2% SDS/PBS. The
samples were subjected to streptavidin enrichment, and on-bead
trypsin digestion according to the published TOP-ABPP protocol.52
The trypsin digest was separated from the beads through 1400g
centrifuge, and the beads were washed with 3 × 300 μL PBS, 3 × 300
μL H2O. The azobenzene cleavage was carried out by incubating the
beads with 3 × 75 μL 25 mM sodium dithionite for 1 h at room
temperature with gentle rotation.53 The supernatant was collected,
added with 5% formic acid and stored at −20 °C until analysis.
For quantification of HNE modified sites, proteomes were treated
with 20 μM and 100 μM HNE separately, labeled with m-APA and
then clicked with heavy biotin-AZO-N3 and light biotin-AZO-N3,
respectively. The proteomes were precipitated with methanol/
chloroform, combined and resuspended in 1.2% SDS/PBS. The
samples were processed by TOP-ABPP protocol as described above.
RD-ABPP for Quantifying Endogenously Carbonylated
Proteins in ferroptosis. 8 × 106 HT1080 cells were seeded in 15
cm dishes to grow overnight and incubated with DMSO (“control”) or
150 nM of RSL3 (“ferroptotic”) for 3 h in serum-free medium. The
cells were collected, lysed by sonication in ice-cold PBS containing
0.5% TritonX-100 and centrifuged at 100 000g for 30 min to remove
cell debris. The protein concentrations were determined by BCA
protein assay and normalized to 2 mg/mL in volume of 1 mL. Lysates
from control or ferroptotic cells were separately labeled by 0.5 mM m-
APA. Proteomes were precipitated with methanol/chloroform and
resuspended in 0.4% SDS/PBS. The samples were added with 1 mM
CuSO4, 100 μM TBTA ligand, 100 μM biotin-(PEG)2-N3, and 1 mM
TCEP to react for 1 h. The proteomes were again precipitated with
methanol/chloroform, resuspended in 1.2% SDS/PBS, diluted to 0.2%
SDS/PBS and subjected to streptavidin enrichment. The enriched
proteins were digested by trypsin on-bead in 100 mM TEAB buffer
and subjected to reductive dimethylation labeling.60,73 Briefly, 4% of
light or heavy formaldehyde was added to ferroptotic or control
proteomes, respectively with 0.6 M sodium cyanoborohydride added
simultaneously. The reaction lasted for 1 h at room temperature and
sequentially quenched by 1% ammonia and 5% formic acid. Finally,
heavy and light labeled peptides were combined, concentrated by
speed vacuum, separated by the Fast-seq protocol,74 and analyzed by
LC-MS/MS.
EXPERIMENTAL SECTION
■
Testing m-APA in a GSH Model. One mM GSH was reacted
with 100 μM HNE in Tris-HCl buffer (pH = 7.4) for 1 h at room
temperature. The reaction was then adjusted to pH 5.0 and added with
0.5 mM m-APA and 60 mM NaBH3CN for another 1 h. The samples
were analyzed on a UPLC-ESI-MS with a peptide BEH C18 column
(Waters 300, 1.7 2.1 100 mm) and a quadrupole rods SQ Detector 2
mass spectrometer (Waters Corp.) The ultrapure water and
acetonitrile are used as the mobile phase in a 7 min gradient, and
the ion chromatographic traces are extracted at m/z 306 for GSH, 462
for GSH-HNE, 563 for GSH-HNE-m-APA.
In-Gel Fluorescence Scanning. H1299 cells were grown to 80%
confluence, washed three times with PBS and centrifuged at 1000 rpm
for 3 min. PBS supernatant were removed and the cell pallets were
stored at −80 °C. The cell pallets were lysed by sonication in ice-cold
PBS containing 0.1% TritonX-100, centrifuged at 100 000g for 30 min
to remove cell debris, and protein concentrations were determined by
BCA protein assay. Proteomes were normalized to 2 mg/mL,
incubated with 0, 10, 50, 100, or 250 μM HNE for 1h at room
temperature and labeled by 0.5 mM m-APA at pH 5.0 for 1 h.
Proteomes were precipitated by adding 5 volumetric times of
methanol/chloroform (v/v = 4:1) mixture and 3 volumetric times of
water, and centrifuged at 10 000g for 10 min. The supernatants were
discarded, and the proteomes were washed using cold methanol and
resuspended in 0.4% (w/v) SDS/PBS. The proteomes were reacted
with 1 mM CuSO4, 100 μM TBTA ligand, 100 μM fluorophore-N3,
and 1 mM TCEP for 1 h at room temperature. The proteomes were
boiled using sample buffer at 90 °C for 5 min, resolved on 10% SDS-
PAGE gels and imaged by ChemiDoc XRS+ (Bio-Rad). The gels were
then stained by Coomassie brilliant blue (CBB) to demonstrate equal
loading.
To check protein carbonylations in ferroptosis by in-gel
fluorescence, HT1080 cells were treated with 0, 50, or 150 nM
RSL3, with or without cotreatment with 300 nM of Liproxstatin-1 for
3 h. Cells were collected, lysed by sonication in ice-cold PBS
containing 0.5% TritonX-100 and centrifuged at 100 000g for 30 min
to remove cell debris. The protein concentrations were determined by
BCA protein assay and normalized to 2 mg/mL. The procedures of m-
APA labeling and in-gel fluorescence scanning were conducted as
described above.
To rescue cells from ferroptosis, HT1080 cells were treated with
150 nM RSL3 together with 300 nM Liproxstatin-1 (“Rescued”).
Proteomes from “ferroptotic” and “rescued” cells were subjected to the
RD-ABPP protocol as described above.
Identification of Endogenous Sites of LDE Modification in
Ferroptosis. For identification of endogenous sites of LDE
modification in ferroptotic cells, HT1080 cells were treated with
DMSO (“control”) or 150 nM of RSL3 (“ferroptotic”) as described
above in RD-ABPP protocol. The proteomes were labeled by 0.5 mM
G
DOI: 10.1021/jacs.8b01462
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
m-APA for 60 min and then reacted with 1 mM CuSO4, 100 μM
TBTA ligand, 100 μM biotin-acid-N3 tag, and 1 mM TCEP for 1 h.
The proteomes were precipitated with methanol/chloroform,
resuspended in 1.2% SDS/PBS, and diluted to 0.2% SDS/PBS. The
samples were subjected to streptavidin enrichment, and on-bead
trypsin digestion. The trypsin digest was separated from the beads
through 1400g centrifugation and the beads were washed with 3 × 300
μL PBS and 3 × 300 μL H2O. The LDE modified peptides were
released by incubating the beads with 2 × 200 μL 2% formic acid for
1h at room temperature with gentle rotation. The beads were washed
with 2 × 400 μL 50% acetonitrile/water containing 1% formic acid,
and the supernatant was combined, concentrated by speed vacuum,
and analyzed by LC-MS/MS.
LC-MS/MS and Data Analysis. Samples were analyzed by LC-
MS/MS on Q Exactive series Orbitrap mass spectrometers (Thermo
Fisher Scientific) coupled with Easynano-LC. Under the positive-ion
mode, full-scan mass spectra were acquired over the m/z range from
350 to 1800 using the Orbitrap mass analyzer with mass resolution of
70 000. MS/MS fragmentation is performed in a data-dependent
mode, of which the TOP 20 most intense ions are selected for MS2
analysis a resolution of 17 500 using collision mode of HCD. Other
important parameters: isolation window, 2.0 m/z units; default charge,
2+; normalized collision energy, 28%; maximum IT, 50 ms; and
dynamic exclusion, 20.0 s.
Present Address
⊥Rudolf Virchow Center for Experimental Biomedicine,
University of Wurzburg, 97080 Wurzburg, Germany.
̈
̈
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Xing Chen and Ms. Pinou Lv for discussion and
help with fluorescence imaging. We thank the Computing
Platform of the Center for Life Science for supporting the
proteomic data analysis. This work was supported by Ministry
of Science and Technology of China (2016YFA0501500),
National Science Foundation of China (21472008, 21521003,
and 81490741), and a “1000 Talents Plan” Young Investigator
Award (C.W.). J.P.F.A. is supported by the Junior Group
Leader program from the Rudolf Virchow Center - University
of Wurzburg.
̈
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AUTHOR INFORMATION
Corresponding Author
*chuwang@pku.edu.cn
ORCID
■
Chu Wang: 0000-0002-6925-1268
H
DOI: 10.1021/jacs.8b01462
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