Imaging and Profiling of Proteins under Oxidative Conditions in Cells and Tissues by Hydrogen-Peroxide-Responsive Labeling

Article abstract of DOI:10.1021/jacs.0c02547

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed a unique chemical tool to elucidate the biological role

Full text of DOI:10.1021/jacs.0c02547

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Article
Imaging and profiling of proteins under oxidative conditions
in cells and tissues by hydrogen-peroxide-responsive labeling
Hao Zhu, Tomonori Tamura, Alma Fujisawa, Yuki Nishikawa, Rong Cheng, Mikiko Takato, and Itaru Hamachi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c02547 • Publication Date (Web): 21 Aug 2020
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Imaging and profiling of proteins under oxidative conditions
in cells and tissues by hydrogen-peroxide-responsive labeling
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Hao Zhu, Tomonori Tamura, Alma Fujisawa, Yuki Nishikawa, Rong Cheng, Mikiko
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1,2
Takato, and Itaru Hamachi*
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
2ERATO, Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-
0075, Japan
Correspondence should be addressed to I.H. (ihamachi@sbchem.kyoto-u.ac.jp)
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Abstract
Reactive oxygen species (ROS) such as hydrogen peroxide (H
2
O ) can inflict damage to
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biomolecules under oxidative stress and also act as signaling molecules at physiological levels.
Here we developed a unique chemical tool to elucidate the biological roles of ROS using both
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fluorescence imaging and conditional proteomics. H
2
2
O -responsive protein labeling reagents
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(Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells
and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with
conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled
proteins using a conditional proteomics workflow. The integrative analysis enabled the
identification of ROS generation and/or accumulation sites with a subcellular resolution. For the
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first time, we characterized that autophagosomes were enriched with H
2
O in activated
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macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative
stress within mitochondria by the conditional proteomics.
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Introduction
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Reactive oxygen species (ROS) are a class of oxygen-containing and reactive molecules that
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are produced in a wide range of biological processes. They are often produced during oxidative
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stress and can damage lipids, proteins, and DNA, which contributes to aging and many human
diseases, including cancer, inflammation, cardiovascular and neurodegenerative diseases.2-5
Evidence also shows that ROS are generated as a normal byproduct of metabolism and at
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physiological levels, can regulate various signaling pathways. In particular, hydrogen peroxide
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(H
2
O
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) exhibits mild reactivity and is relatively stable, making it an ideal signaling molecule
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that can diffuse and travel appreciable distances, while reversibly modifying critical cysteine
thiols for redox signaling.6,10
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H
2
O production in various cell types has been shown to mediate
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diverse physiological processes, ranging from the immune response to growth factor
stimulation.11-14
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Fluorescent sensors that can detect ROS production in living systems are useful tools to
understand their biological functions,10,15-19 as exemplified by the boronate-based probes for the
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detection of H
2
O
.10,15 When coupled with fluorescence microscopy, the localization of ROS
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production in living systems can be visualized. However, such sensors could readily migrate
after ROS sensing by diffusion, which may lead to an inaccurate readout of ROS localization.
Moreover, most fluorescent sensors are not amenable to fixation. Very recently, Chang and co-
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workers described a histochemical approach to detect H
2
O
2
in fixed samples based on puromycin
production. In
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staining;20 but their method did not provide subcellular resolution of H
2
O
2
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addition to these imaging-based methods, redox proteomics that identify the oxidatively
modified proteins is a complementary approach for elucidating the biological roles of ROS.21-26
Despite these advanced technologies, a robust chemical tool capable of integrating multiple
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approaches does not exist, yet is highly desirable for a more comprehensive view of ROS
homeostasis.
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Here we developed a unique chemical probe that allows for both fluorescence imaging and
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profiling of proteins under oxidative conditions in living cells and tissues. We designed H
2
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O -
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responsive labeling reagents named ‘Hyp-L’ that can be activated in the presence of H O to
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covalently tag nearby proteins, producing an immobilized signal compatible with cell/tissue
fixation and subsequent immunostaining. Furthermore, the proteins labeled with Hyp-L can be
enriched and identified using a conditional proteomics workflow (Fig. 1a).27,28 Integrated
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analysis by the Hyp-L-based protein labeling, imaging, and proteomics, together with the H
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O
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imaging by a fluorescent sensor (HYDROP), enabled us to identify autophagosomes as H
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O
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-
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rich vesicular organelles in the activated RAW264.7 macrophages, as well as phagosomes,
endosomes, and lysosomes. We further demonstrated that Hyp-L can be applied to mouse brain
tissues, and the conditional proteomics clearly indicated mitochondrial ROS production upon
inhibition of mitochondrial respiration in hippocampal slices.
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Results
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Design of Hyp-L
Hyp-L utilizes pinacolboronate as an H
2
O
2
-responsive group,10,15 though it is known to also
react with other ROS, e.g., ONOO , for the selective activation under the oxidative conditions
in living systems. The H -mediated boronate oxidation reaction can produce a 2-
−
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2
O
2
2
1
2
fluoromethylphenol intermediate, which undergoes 1,4-elimination to generate a highly
electrophilic quinone methide (QM)30-32 for covalently labeling surrounding proteins. On the
basis of Fick’s law using the calculated half-life of QM, the diffusion radius of the Hyp-L-
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generated QM was estimated to be less than 1 µm in solutions (see the estimation method in the
Supporting Information). Given the crowded microenvironment and the membrane structures in
living cells, the labeling by Hyp-L could be confined within the membrane-enclosed ROS-rich
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organelles. Two types of Hyp-L, Hyp-L-1 and Hyp-L-2, were prepared by tethering coumarin
and diacetyl fluorescein, respectively, for fluorescent imaging and/or affinity tagging of the
labeled proteins.
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Figure 1 a) Illustration of Hyp-L-based conditional protein labeling for profiling and imaging of
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proteins under the oxidative conditions in cells and tissues. b) Schematic of H
2
2
O -responsive
protein labeling by Hyp-L. WB: western blot, Nu: nucleophilic amino acid, PinB:
pinacolboronate.
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H
2
O
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-modulated reactivity of Hyp-L
The reactivity of Hyp-L-1 to H was evaluated by HPLC. Upon the addition of H
2
O
2
2
O , Hyp-
2
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L-1 and its boronic-acid (BA) derivative (pinacolboronate was partially decomposed to BA
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during HPLC ) were rapidly consumed (Fig. 2a and Supplementary Fig. 1). Simultaneously, a
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hydroxymethylphenol derivative, the hydrolyzed product of QM, was newly generated as a
major product with a recovery yield of ~80% after 30 min. A dimer formed by conjugation of
one QM with one hydrolyzed QM was also detected with a quite low yield, suggesting the self-
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labeling of Hyp-L-1 was neglected. The H
2
O -modulated hydrolysis proceeded in a dose-
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dependent manner (Supplementary Fig. 2).
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The H
2
O
2
-responsive protein labeling was then examined using bovine serum albumin (BSA)
was added to a solution of BSA containing Hyp-L-1, and the
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as a model substrate in vitro. H
2
O
2
1
reaction was analyzed by SDS-PAGE and in-gel fluorescence imaging. The fluorescent BSA
band appeared at 2 min and its intensity increased until 30 min (Supplementary Fig. 3a). A
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dramatic increase in BSA labeling was elicited by 50 µM of H
(Supplementary Fig. 3b). By contrast, no fluorescent bands appeared in the absence of H
clearly indicating that H -responsive BSA labeling had occurred. We also confirmed that Hyp-
2
O
2
(1 eq. to Hyp-L-1)
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2
2
O ,
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2
O
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L-1 was moderately selective for H
2
O
2
compared with other ROS and reactive nitrogen species
(RNS), including O −
, O
1
−
−
1
2
2
, ×OH, ClO , and ×NO, except for ONOO which was also able to
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trigger the BSA labeling (Fig. 2b and 2c). The labeling occurred at physiological pH, and the rate
was moderately enhanced under basic conditions (between pH 6.0–8.0, see Supplementary Fig.
3c), likely due to the increased reactivity of nucleophilic amino acids of BSA upon deprotonation
at basic pH.
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The labeled amino acids were then identified by a conventional peptide mapping protocol,
which included protein digestion and LC-MS/MS analysis. Eight types of nucleophilic amino
acids were modified with Hyp-L-1, including His, Lys, Cys, Glu, Tyr, Ser, Thr, and Asp,
whereas less reactive amino acids, such as Gly, were not labeled (Fig. 2d, Supplementary Fig. 4
and Fig. 5, and Supplementary Data 1). The labeled residues were primarily positioned and
broadly distributed on the surface of BSA. This result revealed the high reactivity and wide
amino acid coverage of the Hyp-L-generated QM.
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Figure 2 Activation of Hyp-L-1 by H
2
O
2
. a) Time course of hydrolysis in the presence of H
2
O .
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50 µM of Hyp-L-1 was incubated with 500 µM of H
2
O
2
in PBS (−). The percentages of Hyp-L-1
and hydrolyzed QM were calculated according to the HPLC peak areas in Supplementary Fig. 1.
Error bar indicates s.d., n = 3. b) ROS selectivity in BSA labeling. 200 µM of various ROS was
added to a PBS (−) solution of BSA (10 µM) containing 50 µM of Hyp-L-1, and the mixture was
incubated for 30 min. The upper panel shows a Coomassie Brilliant Blue (CBB) stained gel
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image. c) Quantification of the labeling in b). The labeling yields were calculated based on the
in-gel fluorescence intensities of Hyp-L-1-labeled BSA and a standard Dc-BSA (see the
calculation method in the Supporting Information). Statistical analyses were performed with a
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two-tailed Student’s t-test (n = 3) relative to the data of H
2
2
O -induced labeling. *P < 0.05, n.s.:
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not statistically significant. d) Hyp-L-1-modified amino acid residues on BSA.
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H
2
O
2
-responsive labeling in living cells
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For the labeling in living cells, we used Hyp-L-2 bearing diacetyl fluorescein as a reporter,
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because i) the fluorescein dye emits at a longer wavelength than coumarin, ii) its antibody is
available with a high and specific affinity, and iii) masking with two acetyl groups, which can be
readily cleaved by intracellular esterases, greatly improves cell permeability. RAW264.7
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2
1
macrophage was selected as a cell line for H
2
O
-mediated immune response assays.16,35,36 CLSM
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imaging showed that Hyp-L-2 could be rapidly taken up by living cells within 5 min, and bright
intracellular fluorescence was broadly distributed across the entire cell (Supplementary Fig. 6),
which is preferable for unbiased intracellular protein labeling. Hyp-L-2 showed low cytotoxicity
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after the incubation for 1 h (Supplementary Fig. 7). The H
living cells was initially tested by adding exogenous H
fluorescent bands over a wide molecular weight range were clearly detected by in-gel
fluorescence imaging. However, in the absence of H , no significant signals were observed
(Fig. 3a and 3b). The increased fluorescence bands were detectable at 10 µM H , suggesting
sufficient sensitivity of Hyp-L-2 to physiological levels of H
.8,9 The labeled proteins could be
2
O
2
-responsive labeling of Hyp-L-2 in
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2
O
2
. In the presence of H , multiple
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O
2
2
O
2
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O
2
2
1
2
2
O
2
2
directly visualized by CLSM imaging in fixed cells, revealing that the fluorescein-tagged
proteins were intracellularly localized with an unbiased distribution (Fig. 3c). By contrast, non-
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H
2
O
2
-treated cells became relatively dark after cell fixation, because the small molecules of
unreacted Hyp-L-2 could be easily washed out. As a control, we used HYDROP, a commercially
available fluorescent H sensor. Its fluorescence clearly increased upon H addition in living
cells (Supplementary Fig. 8) but was extinguished after cell fixation (Fig. 3d). This highlighted a
key advantage of Hyp-L-2 over conventional H sensors for fixed-cell imaging.
2
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O
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Figure 3 H
SDS-PAGE analysis. Cells were treated with 5 µM of Hyp-L-2 for 10 min, followed by
incubation with a varied concentration of H for 30 min. b) Quantification of the labeling in a).
The band intensities were normalized to the data of H (-). Error bar indicates s.d., n = 3.
2
O
2
-responsive protein labeling by Hyp-L-2 in living RAW264.7 macrophages. a)
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O
2
2
O
2
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CLSM imaging of the fixed cells after the labeling by Hyp-L-2 (c) and HYDROP (d). H
2
2
O : 100
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µM. The cells were fixed with chilled methanol. Scale bars, 10 µm.
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Conditional protein labeling in PMA-activated macrophages
2
We next tested Hyp-L-2 in the conditional protein labeling upon physiological stimulation.
Professional phagocytes, such as macrophages, produce ROS during immune response when
phagocytosis of microbes or encountering non-particulate stimuli. Phorbol myristate acetate
(PMA) is one such stimulus and mimics the signal transduction pathway that assembles NADPH
oxidase (NOX).36-39 Diverse ROS-rich vesicles have been discovered in PMA-treated RAW264.7
macrophages by fluorescence imaging using ROS sensors.16,35 However, the exact nature of
these ROS-producing/accumulated organelles has not yet been determined.37-39 We thus
attempted to address this by identifying proteins in the ROS-rich vesicles using the Hyp-L-based
proteomics.
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We initially evaluated the protein labeling by Hyp-L-2 in response to PMA stimulation in
RAW264.7 macrophages. Cells were treated with PMA for 30 min, followed by incubation with
Hyp-L-2 for another 30 min. Protein labeling was assessed by western blot using an anti-
fluorescein antibody (Fig. 4a and 4b). The band intensity and the number of labeled proteins
substantially increased upon the PMA stimulation, and this could be blocked by inhibition of
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NOX with diphenyleneiodonium (DPI) or apocynin. Ebselen, a mimic of the H
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O scavenger
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glutathione peroxidase, and N-acetylcysteine (NAC), a frequently used antioxidant, also
significantly quenched the labeling. L-NAME can inhibit the production of ×NO by nitric oxide
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synthase (NOS) and the subsequent conversion of ×NO to ONOO . We found that L-NAME had
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little effect on the protein labeling and the expression of inducible NOS (iNOS) kept minimal
upon the PMA stimulation (Supplementary Fig. 9). These results indicate that the endogenously
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could be mainly responsible for the enhanced protein labeling by Hyp-L-2 in
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PMA-stimulated macrophages. By contrast, a bromo analogue of Hyp-L-2, compound 8, lacking
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the H
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-sensitive boronate group, was unable to produce any protein labeling signals regardless
of the PMA stimulation (Supplementary Fig. 10).
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We next visualized the labeled proteins by CLSM imaging after the cell fixation. Strong and
punctate fluorescence from vesicles was observed in the PMA-treated cells, whereas the
untreated control cells showed negligible fluorescence (Fig. 4c). Notably, PMA did not affect the
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cellular uptake of Hyp-L-2 (Fig. 4d). This might suggest that the elevated intracellular H
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likely enclosed within the vesicular organelles. The punctate fluorescence was abolished by the
treatment with DPI, apocynin, ebselen, and NAC, but not affected by L-NAME (Supplementary
Fig. 11), which was consistent with the above western blot data (Fig. 4a and 4b). We also
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Figure 4 Conditional protein labeling in RAW264.7 macrophages upon PMA stimulation. a)
Western blot analysis. RAW264.7 macrophages were activated with 1 µg/mL of PMA in the
absence or presence of various inhibitors or scavengers for 30 min, followed by incubation with
5 µM of Hyp-L-2 for 30 min. After cell lysis and SDS-PAGE, the labeled proteins were detected
by western blot using an anti-fluorescein antibody. DPI: 10 µM, apocynin: 5 mM, L-NAME: 5
mM, ebselen: 5 µM, NAC: 10 mM. b) Quantification of the labeling in a). The band intensities
were normalized to the data of PMA (+). Statistical analyses were performed with a two-tailed
Student’s t-test (n = 3) relative to the data of PMA (+). *P < 0.05, **P < 0.01, ***P < 0.01, n.s.:
not statistically significant. CLSM imaging of the fixed (c) and living (d) cells labeled by Hyp-L-
2. After the PMA stimulation as a), the cells were incubated with Hyp-L-2 for 30 min (c) or 10
min (d). The cells were fixed with chilled methanol in c) before imaging. Scale bars: 10 µm.
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Proteomic profiling and vesicle identification
To identify proteins associated with the H O produced upon PMA stimulation, we coupled the
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labeling by Hyp-L-2 with LC-MS/MS analysis (Fig. 5a). The labeled proteins were enriched by
immunoprecipitation (IP) (Fig. 5b) and in-gel digested by trypsin. We carried out quantitative
mass analysis using tandem mass tagging (TMT). The digested peptides were tagged by heavy
(H) and light (L) TMT reagents for PMA (+) and PMA (−) samples, respectively, which were
then mixed in a 1:1 ratio and subjected to LC-MS/MS analysis.
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A total of 171 proteins were identified, among which 170 (> 99%) proteins showed above zero
values in log2 fold change (H/L ratio), indicating that the protein labeling by Hyp-L-2 was
indeed strongly affected by PMA stimulation (Fig. 5c and Supplementary Data 2). Six proteins,
such as Eif4a1, Rab5c, and Pfn1, were only labeled in PMA-treated macrophages with a
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maximal H/L ratio of 100, and 63 proteins were significantly enriched (log (H/L) > 1) with high
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fidelity (P < 0.05). These 69 promising protein candidates were categorized into group A for
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further analysis.
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To characterize the labeled proteins in group A, we performed gene ontology (GO) enrichment
analysis. The cellular component analysis showed that vesicular proteins were significantly
enriched (Fig. 5d), consistent with the punctate signals in our fixed-cell imaging. We also
conducted western blot analysis of several highly-enriched proteins in group A (Supplementary
Fig. 14), as well as DNA microarray analysis for the whole transcriptome in RAW264.7
macrophages (Supplementary Fig. 15 and 16). These results indicated that expression at both the
protein and mRNA levels did not change significantly before and after the PMA stimulation.
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Thus, the proteomic profiling most likely reflected responsive protein labeling in the H
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compartments, rather than changes in protein expressions.
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We next attempted to identify the H
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proteins in group A could be assigned to specific vesicular organelles, including phagosomes,
endosomes, and lysosomes (Supplementary Table 1). This is consistent with the previous reports
that phagosomes and endosomes recruit NOX on their membranes and enrich ROS during the
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immune response.1,38,39,41 Also, it is known that overproduced H
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can diffuse into lysosomes.42
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The HYDROP-stained vesicles in PMA-treated living cells indeed moderately colocalized with
LysoTracker Deep Red (LysoDR, Supplementary Fig. 17), a fluorescent dye for staining acidic
organelles, such as lysosomes and endosomes. We also noticed that GO enrichment analysis of
proteins in group A characterized intracellular compartments other than vesicles, such as
ribosomes and mitochondria (Fig. 5d). We selected two mitochondrial proteins in group A, Sdha
(H/L ratio = 3.39) and Hspa9 (H/L ratio = 2.77), for immunostaining after the conditional
labeling by Hyp-L-2. The antibodies were validated by siRNA knockdown (Supplementary Fig.
18 and Fig. 19). Sdha and Hspa9 showed punctate signals in the PMA-activated macrophages,
which could be partially merged with the Hyp-L-2-labeled vesicles (Fig. 5e and Supplementary
Fig. 20). Given previous studies,43,44 these might be caused by a self-engulfing process inside
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cells via PMA-induced autophagy. We therefore hypothesized that H
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within autophagosomes. Indeed, HYDROP was partially colocalized with mCherry-LC3B, a
standard autophagosome marker (Fig. 5f and Supplementary Fig. 23). Taken together, we
concluded that the H
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O generated upon PMA stimulation in RAW264.7 macrophages was likely
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enriched within a wide range of vesicular organelles, including phagosomes, endosomes,
lysosomes, and autophagosomes.
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Figure 5 Proteomic analysis of the labeled proteins in RAW264.7 macrophages. a) Workflow
for TMT-based quantitative proteomic analysis of PMA (+) vs PMA (-). b) In-gel fluorescence
imaging of IP-enriched proteins. c) Volcano plot obtained by the P-value of H: PMA (+) vs L:
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PMA (-) against Log
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(H/L). The proteins showing log (H/L) > 1.0 and P < 0.05 were
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highlighted in red. P-value was calculated by the Benjamini–Hochberg procedure. Six proteins
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only labeled in the PMA (+) sample were not plotted, including Eif4a1, Rab5c, Pcbp1, Pfn1,
Ppl27a, and Lmnb1. Two proteins analyzed by immunostaining were denoted. d) GO enrichment
analysis of the 69 proteins in group A (64 protein IDs were mapped). e) Representative CLSM
imaging of the immunofluorescence against Sdha that were partially merged with Hyp-L-2-
labeled vesicles in PMA-stimulated RAW264.7 macrophages. The nucleus was stained with
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Hoechst 33258 (Ho258). The antibody was validated by siRNA knockdown (Supplementary Fig.
18 and Fig. 19). f) Representative CLSM imaging of mCherry-LC3B with HYDROP in PMA-
stimulated RAW264.7 macrophages. Images from 4 separate fields containing over 10 cells were
collected and analyzed for e) and f). See Supplementary Fig. 20 and Fig. 23 for images in other
fields. Line-plot graphs indicate the fluorescence intensity profiles of Sdha or LC3B (red) and
Hyp-L-2 (green) along the white dotted lines. Colocalization was quantified using Pearson’s r.
Scale bars, 5 µm and 2 µm (enlarged images).
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Conditional protein labeling under mitochondrial oxidative stress
10
Hyp-L-2 is applicable for the conditional protein labeling within other cellular compartments.
Mitochondrial respiratory chain is the main producer of cellular energy and is also a major
source of ROS in mammalian cells. Disorder of the mitochondrial respiratory chain complexes
may therefore promote the overproduction of ROS. We used 2-thenoyltrifluoroacetone (TTFA),
a mitochondrial complex II inhibitor that binds to the ubiquinone-binding site, to induce
oxidative stress within mitochondria in HeLa cells.45,46 SDS-PAGE analysis showed increased
protein labeling by Hyp-L-2 in response to the TTFA stimulation (Fig. 6a and 6b). To determine
the subcellular localization of the labeled proteins, HeLa cells were stained with MitoTracker
Deep Red (MitoDR) prior to the treatment with TTFA and labeling by Hyp-L-2. MitoDR
contains a thiol-reactive chloromethyl moiety for mitochondrial labeling, that allows imaging of
fixed cells. Although Hyp-L-2 inside living cells treated with TTFA was homogenously
distributed throughout the cell, the retained Hyp-L-2 fluorescence after cell fixation was
heterogeneous (Fig. 6c) and merged well with MitoDR (Fig. 6d). This gave us additional
evidence that the Hyp-L-based conditional labeling was able to address the ROS production with
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a subcellular resolution. Interestingly, we observed TTFA-induced fragmentation of
mitochondria into punctate vesicles (Supplementary Fig. 24), which is consistent with the
previous report that inhibition of mitochondrial respiratory chain causes ROS-mediated
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autophagy.46
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Figure 6 Conditional protein labeling under mitochondrial oxidative stress in HeLa cells. a)
SDS-PAGE analysis of TTFA-induced labeling. HeLa cells were treated with 1 mM of TTFA for
30 min, followed by incubation with 5 µM of Hyp-L-2 for 30 min. b) Quantification of the
labeling in a). The band intensities were normalized to the data of TTFA (-). Statistical analyses
were performed with a two-tailed Student’s t-test (n = 4) relative to the data of TTFA (-). **P <
0.01. c) CLSM imaging of TTFA-treated cells. After the labeling as a), cells were subjected to
CLSM imaging before (upper) and after (lower) the fixation with chilled methanol. d)
Colocalization imaging of Hyp-L-2 with MitoDR in TTFA-treated cells. HeLa cells were stained
with 200 nM of MitoDR before the TTFA stimulation and the Hyp-L-2 labeling. The cells were
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fixed with chilled methanol before CLSM imaging. Colocalization was quantified using
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Application of Hyp-L to mouse brain tissue
5
The brain is a metabolically active organ exhibiting a high demand for energy and oxygen,
together with robust ROS production. Abnormally high levels of ROS in the brain lead to
neuronal toxicity and neurodegeneration,3 on the other hand, ROS at physiological
concentrations are involved in synaptic plasticity.47,48 We further explored the applicability of
Hyp-L in mouse brain tissues. Acute hippocampal slices (250 µm thick) were prepared and
incubated with Hyp-L-2 in artificial cerebrospinal fluid (ACSF) for 60 min, followed by
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treatment with 1 mM of H
signal in response to H
25a). Z-axis scanning of the labeled slices revealed efficient tissue penetration of Hyp-L-2 to a
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for 30 min and tissue fixation. An increase in the fluorescent
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was clearly observed by fixed-tissue imaging (Supplementary Fig.
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depth of ~100 µm (Supplementary Fig. 25b). Western blot analysis confirmed the H
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protein labeling by Hyp-L-2 in the tissues (Supplementary Fig. 26).
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Conditional protein labeling in brain tissue was demonstrated under the oxidative stress
induced by TTFA. SDS-PAGE analysis showed significantly increased fluorescent bands in
hippocampal slices treated with TTFA, which was suppressed by ebselen or skQ1, a
mitochondrial antioxidant (Fig. 7a and 7b). CLSM imaging with HYDROP in living slices
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verified the increased H
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2
O production induced by TTFA (Supplementary Fig. 27). However,
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such fluorescence signals were completely abolished after tissue fixation (Fig. 7c). By contrast,
TTFA-induced labeling by Hyp-L-2 remained fluorescent in the fixed slices (Fig. 7d and 7e). We
subsequently attempted to investigate whether the conditional labeling indeed occurred within
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mitochondria via colocalization imaging by using MitoDR and anti-Sdha, a mitochondrial
antibody marker. After fixation and permeabilization, the slices were further trimmed to 40 µm
to improve antibody penetration. Unfortunately, the CLSM imaging results were ambiguous
(Supplementary Fig. 28), because the complex tissue structure made it difficult to distinctly
recognize mitochondria, unlike that in simple cultured cells.
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Figure 7 Application of Hyp-L-2 to mouse brain tissues. a) SDS-PAGE analysis of the
conditional protein labeling upon TTFA stimulation. Acutely prepared slices were treated with 1
mM of TTFA in the absence or presence of ebselen or skQ1 (10 µM) for 30 min, followed by
incubation with 5 µM of Hyp-L-2 for 60 min. The right panel shows an image of the Ponceau S
stained PVDF membrane. b) Quantification of the labeling in a). The band intensities were
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normalized to the data of TTFA (+). Statistical analyses were performed with a two-tailed
Student’s t-test (n = 3) relative to the data of TTFA (+). *P < 0.05, **P < 0.01. CLSM imaging
of hippocampal slices labeled by HYDROP (c) and Hyp-L-2 (d) in response to the TTFA
stimulation. After the labeling as a), slices were fixed, permeabilized, and subjected to CLSM
imaging. Scale bars, 500 µm. e) Quantification of the labeling in c) and d). The fluorescence
intensities were extracted from seven POIs in each image of c) and d). Statistical analyses were
performed with a two-tailed Student’s t-test (n = 7) relative to the data of TTFA (-). **P < 0.01,
n.s.: not statistically significant.
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As demonstrated above, proteomics data are complementary to imaging experiments by
providing valuable information about the subcellular sources of ROS. We thus used TMT-based
quantitative proteomics to clarify the localization of the labeled proteins (Fig. 8a). A total of 253
proteins were identified, which included 252 proteins showing above zero log2 fold change (H:
TTFA (+)/ L: TTFA (−)) values (Fig. 8c and Supplementary Data 4). 130 proteins that were
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highly and significantly enriched (log (H/L) > 1, P < 0.05) were categorized into group B and
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subjected to GO enrichment analysis. The cellular component analysis revealed that the labeled
proteins were selectively and significantly enriched in mitochondria, while some proteins were
moderately enriched in the cytosol and cytoskeleton (Fig. 8d). Biological process analysis
significantly characterized ROS- and mitochondria-associated processes, such as ROS metabolic
processes and electron transport chain (Fig. 8e), in good agreement with the cellular component
analysis. The proteins in group B were also individually analyzed with Uniprot, identifying 35
mitochondrial proteins, including 8 proteins assigned to the mitochondrial respiratory chain
(Supplementary Data 4). In particular, 7 of the top 10 proteins in group B were annotated as
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mitochondrial proteins (Fig. 8c). These results clearly demonstrated that the Hyp-L-based
conditional proteomics worked well even in tissue samples and that TTFA indeed
pharmacologically enhanced ROS production in mitochondria of hippocampal slices.
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Figure 8 Proteomic analysis of the labeled proteins in hippocampal slices. a) Workflow for
TMT-based quantitative proteomic analysis of TTFA (+) vs TTFA (−). b) In-gel fluorescence
imaging of IP-enriched proteins. c) Volcano plot obtained by the P-value of H: TTFA (+) vs L:
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TTFA (−) against Log
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(H/L). The proteins showing log (H/L) > 1.0 and P < 0.05 are
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top 10 proteins in group B that assigned to mitochondria were denoted. d) Cellular component
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and e) biological process analysis of the 130 proteins in group B (111 protein IDs were mapped).
Nu: nucleus, Mt: mitochondrion, ER: endoplasmic reticulum, PM: plasma membrane.
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Discussion
5
We have developed a new chemical probe, Hyp-L, which relies on H
2
2
O -induced covalent
6
protein labeling in live cells, for both fluorescence imaging and profiling of proteins under the
oxidative conditions. It was further demonstrated that the Hyp-L-based conditional proteomics
was able to identify oxidative stress with a subcellular resolution in tissue specimens, which
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remains challenging for imaging with the conventional H
2
2
O sensors due to the complex
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structure and cell-type heterogeneity of tissues.
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Although useful, it is noteworthy to point out the (potential) limitations and drawbacks of Hyp-
L, including (i) the moderate ROS selectivity, (ii) the relatively large diffusion radius, (iii) the
pH dependence on the labeling reaction, (iv) the potential competition and/or consumption by
cellular antioxidants, (v) potentially biased protein labeling due to the limited amino acid
coverage and/or interactions with some specific proteins. Given these, one should carefully
design the experiments and analyze/validate the results provided by Hyp-L. For instance,
inhibitory assays for ROS/RNS generation enzymes may determine whether the conditional
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labeling by Hyp-L is due to H
2
O
2
. The production and distribution of H
2
2
O can be validated by
the imaging with some H
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O
2
-selective sensors.16,18 Also, characterizing proteins that may
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localize in subcellular compartments of acidic pH and/or rich cellular antioxidants must be
conducted with great attention.
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Indeed, such integrative analysis that was triggered with experiments of Hyp-L provided a
wealth of data regarding the subcellularly resolved ROS homeostasis as shown here. We
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identified, for the first time, that autophagosomes contained a high level of H
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O
2
in the activated
macrophages. Our results provide direct evidence of the linkage between H
2
O
2
homeostasis and
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autophagy, which will be valuable for elucidating the mechanism underlying the ROS-induced
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autophagy in immunity.50
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We envision the future use of Hyp-L to unravel important, yet to be clearly addressed, issues
regarding ROS roles in diverse physiological and pathological processes, such as NMDR
receptor-mediated signaling,48 inflammation, and ischemia-reperfusion injury. For these
directions, the further improvement of probe molecules is keenly required in several issues
including the points mentioned above, as well as the development of various chemical probes
showing distinct ROS selectivity.
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Supporting information
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Supplementary figures and tables, experimental procedures, synthesis, and characterization of
compounds; Supplementary Data 1-4.
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Acknowledgments
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We thank Dr. Shigeki Kiyonaka (Nagoya University) for support in brain slice experiments, Dr.
Wataru Aoki (Kyoto University) for experimental support in the determination of labeling sites,
and Dr. Takayuki Miki (Tokyo Institute of Technology) for discussions in probe design and
proteomic analysis. We also thank Kathy Tamai in Edanz Group (www.edanzediting.com) for
editing a draft of this manuscript. This work was funded by Grant-in-Aid for JSPS fellows
(17F17344) to H.Z., Grant-in-Aid for Young Scientists (18K14334) and Grant-in-Aid for
Scientific Research on Innovative Areas “Integrated Bio-metal Science” (19H05764) to T.T., and
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Japan Science and Technology Agency (JST) ERATO Grant JPMJER1802 to I.H. This work
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was also supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemistry
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for Multimolecular Crowding Biosystems” (17H06348) to I.H.
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Competing financial interests
The authors declare no competing interests.
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