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A set of organelle-localizable reactive molecules for mitochondrial chemical proteomics in living cells and brain tissues Yuki Yasueda, Tomonori Tamura, Alma Fujisawa, Keiko Kuwata, Shinya Tsukiji, Shigeki Kiyonaka, and Itaru Hamachi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b02254 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016
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A set of organelle-localizable reactive molecules for mitochondrial chemical proteomics in living cells and brain tissues
4
Yuki Yasueda,1 Tomonori Tamura,1 Alma Fujisawa,1 Keiko Kuwata,2 Shinya Tsukiji,3,4
5
Shigeki Kiyonaka,1 and Itaru Hamachi1,5
1 2
6 7
1
8
Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, JAPAN
9
2
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of
Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Chikusa, Nagoya,
10
464-8602, JAPAN
11
3
12
Showa-ku, Nagoya, 466-8555, JAPAN
13
4
14
Showa-ku, Nagoya, 466-8555, JAPAN
15
5
16
Chiyodaku, Tokyo, 102-0075, JAPAN
Frontier Research Institute for Materials Science, Nagoya Institute of Technology, Gokiso,
Department of Materials Science and Engineering, Nagoya Institute of Technology, Gokiso,
CREST(Core Research for Evolutional Science and Technology, JST), Sanbancho,
17 18
Correspondence should be addressed to I.H. (
[email protected]).
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Abstract
2
Protein functions are tightly regulated by their subcellular localization in live cells and
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quantitative evaluation of dynamically altered proteomes in each organelle should provide
4
valuable information. Here, we describe a novel method for organelle-focused chemical
5
proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive
6
molecules (MRMs) were designed that penetrate biomembranes and spontaneously
7
concentrate in mitochondria, where protein labeling is facilitated by the condensation effect.
8
The combination of this selective labeling and liquid chromatography–mass spectrometry
9
(LC-MS) based proteomics technology facilitated identification of mitochondrial proteomes
10
and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live
11
cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of
12
mitochondrial
13
oxidant-induced apoptotic process was performed by combination of this MRMs-based
14
method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino
15
acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical
16
proteomics to elucidate mitochondria-associated biological events and diseases.
proteins
whose
expression
levels
change
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significantly
during
an
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Introduction
2
Proteome analysis relying on advanced mass (MS) spectroscopy is now a powerful
3
strategy for large scale profiling of proteins in biological samples, including cell lysates, live
4
cells and in vivo.1 The combination of chemical protein labeling with MS-based proteome
5
techniques has remarkably facilitated the profiling capability, by defining a specific class of
6
proteins with labeling selectivity.2 One of the most successful examples represents so-called
7
activity-based protein profiling (ABPP), which was invented by Cravatt’s group.3 Here, only
8
proteins exhibiting an enzymatic activity-of-interest can be labeled with a bio-orthogonal
9
handle. ABPP employs active site-directed chemical reagents (known as activity-based
10
probes: ABPs) to label proteins with a particular enzyme activity in their active pocket, which
11
can focus on the proteome depending on a particular functional state of enzymes, prior to
12
MS-fingerprinting analysis.
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In addition to activity, protein localization in a variety of subcellular compartments
14
of live cells should be crucial for proteomics, because the expression level and localization of
15
proteins in each organelle are dynamically varied in response to environmental conditions and
16
tightly involved in their biological functions. The spatio-temporal changes of proteomes in
17
organelles are responsible for the healthy state of organelles, cells and the consequent live
18
bodies. For example, mitochondria, an energy generating organelle, plays crucial roles in
19
regulating redox states of whole cells and mitochondrial dysfunction induced by a perturbed
20
proteome is associated closely with serious diseases,4 such as cancer, Alzheimer’s and
21
Parkinson’s diseases. Therefore, it is crucial to explore what proteins are expressed within a
22
specific organelle and how the proteome changes dynamically with a particular stimulus,
23
thereby addressing a number of fundamental questions that should facilitate medical 3 ACS Paragon Plus Environment
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applications.
2
In conventional organelle-focused proteomics, subcellular fractionation followed by
3
a high-resolution 2D-gel and/or LC-MS/MS is a widely accepted approach to characterize
4
organelle proteome.5 However, despite careful preparation, the isolated organelle of interest
5
often suffers from contamination of other organelles and/or potential loss of significant
6
components, which leads to the false-positive and false-negative results. A new method that
7
allows the organelle-focused proteomic mapping without relying on any fractionation was
8
developed recently by Ting and coworkers.6 They selectively over-expressed an
9
organelle-targeting engineered peroxidase enzyme in mitochondria, by which spatially limited
10
oxidation reactions were facilitated to selectively modify mitochondrial proteins with a biotin
11
affinity tag, followed by extensive MS analysis. Although promising, this enzymatic method
12
always requires expression of an engineered peroxidase. Hence it is difficult to apply the
13
technique to cells or tissues7 (such as primary neurons and brain slices) that are not readily
14
amenable to exogenous gene expression.8 In addition, this method uses rather harsh reaction
15
conditions (a high concentration of H2O2), which may detrimentally perturb natural biological
16
environments9 and such phenoxyl radical-mediated labeling may be unsuited for proteins that
17
lack accessible electron-rich residues such as tyrosine on their surface.6 To sidestep these
18
drawbacks, chemistry-based proteomics focusing on an organelle would be attractive.
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Recently, a few of organelle-directed ABPs have been reported, in which organelle-targeting
20
moieties are conjugated with ABPs.10 However, the ABPs that label a limited enzyme family
21
may not well cover broad-range of proteomes in a target-organelle. For the comprehensive
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organelle-proteomics, novel approaches not relying on a particular enzyme activity should be
23
required. Toward this end, we recently reported nuclear-localizable reactive molecules 4 ACS Paragon Plus Environment
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(NRMs) containing a DNA-binding motif and a reactive chloroacetyl group.11 The reagents
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spontaneously accumulated in the nucleus and selectively reacted with nuclear proteins under
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intact cell conditions, which allowed the characterization of a range of nuclear proteins by
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combination with LC-MS/MS analysis.
5
We describe herein a novel chemical tool for mitochondria-focused proteomics that
6
exploits a set of mitochondria-localizable reactive molecules (MRMs) exhibiting a variety of
7
chemical reactivity and selectivity for endogenous proteins. Spontaneous concentration and
8
the resultant high concentration of MRMs in mitochondria allowed for spatially restricted
9
chemical reactions with natural mitochondrial proteins of live cells. By combination of this
10
mitochondria-selective labeling with MS-based protein identification, many labeled peptides
11
can be identified. Thus, the chemo-selectivity of these reactive moieties toward a broad array
12
of side chains of endogenous proteins in live cells can be addressed in detail. Owing to its
13
mild labeling conditions and simple protocol, which need neither genetic manipulation nor
14
organelle fractionation, we succeeded in profiling many mitochondrial proteins in live primary
15
cultured neurons and even in brain slices, as well as HeLa cell lines. Furthermore, coupling of
16
this method with a standard quantitative MS technique (SILAC: stable isotope labeling by
17
amino acids in cell culture) enabled to analyze the dynamic change of mitochondrial proteome
18
induced by an oxidative stress.
19 20
Results and discussion
21
Strategy and molecular designs of mitochondria-localizable reactive molecules (MRMs).
22
For analyzing the mitochondrial proteome, a set of chemically reactive small molecules
23
equipped with mitochondria-targetable function were designed (Figure 1). Since there are 5 ACS Paragon Plus Environment
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various nucleophilic amino acid residues exposed on the surface of proteins, many
2
electrophilic molecules can potentially react to modify proteins.12,17 Therefore, it was expected
3
that chemical labeling of proteins located in mitochondria should be facilitated by
4
co-localization of MRMs with mitochondrial proteins, when these are efficiently condensed
5
together in the same small spaces like the case of NRMs.11 Proteins modified by such spatially
6
limited reactions are then analyzed and identified after cell lysis to collect data on their
7
localization in live cells. That is, MRMs-based labeling allows the transfer of dynamically
8
altered information on mitochodrial proteome in live cells to a permanent covalent bond.
9
We prepared MRMs composed of both a mitochondria-localizing moiety and a
10
chemically reactive moiety. Tetraethyl-rhodamine (Et4-Rhod) was employed as the
11
localizable moiety for mitochondria13 so that the localization of the MRMs can be readily
12
evaluated by live-cell fluorescent imaging. As a reactive moiety, on the other hand, a variety
13
of reactive functionalities exhibiting distinct amino acid selectivity were used, with the aim of
14
modifying as many amino acids as possible.14 We explored eight reactive moieties here.
15
Among them, chloroacetyl (CA) and maleimide (MI) are expected to preferentially react with
16
the thiol group of cysteines.14a, 15 Amino groups such as the α-amine at the N-terminus of
17
proteins and the ε-amine of lysine side chains are reactive with thiophenyl ester (TE) and
18
thiazolidinethione (TZ), which gives a stable amide bond.16 Epoxide (EP) is reported to react
19
with various nucleophilic amino acids, such as Cys, Lys and His.17 Sulfonyl fluoride (SF) was
20
used previously to label active sites of Ser proteases as ABPP probes and was also expected to
21
react with Lys and Tyr residues.18 To label a wider range of amino acids, photoreactive groups
22
such as phenylazide (PA) and benzophenone (BP), conventional photoaffinity labeling
23
reagents, were also employed.19 Figure 1 summarizes a set of such MRMs and their 6 ACS Paragon Plus Environment
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(expected) amino acid preferences. Synthesis and characterization of the derivatives by NMR
2
and MS spectroscopy was conducted according to Scheme S1.
3 Living Cell
Mitochondria
LC-MS/MS m/z m/z
Mitochondria-selective protein labeling
Identification of ! Mitochondrial proteome
Mitochondria-localizable ! Reactive Molecules (MRMs)
Localizable moiety!
Reactive moiety
O O
N
Cl
N+
O
O
O N
1b(EP)
Cys
Cys, Lys, His
F
O
N H
O
1a(CA)
O S
O
O
S O
1c(SF)
1d(TE)
Ser, Lys, Tyr
Lys
N O
Et4-Rhod
O N O
N S
5 6 7 8
O
O
1e(TZ)
4
O
N3
S
1f(BP)
Lys
1g(PA)
O
O
1h(MI) Cys
Figure 1. Schematic illustration of mitochondria selective protein labeling and profiling by a set of mitochondria-localizable reactive molecules (MRMs). Et4-Rhod, tetraethyl-rhodamine; CA, chloroacetyl; EP, epoxide; SF, sulfonyl fluoride; TE, thiophenyl ester; TZ, thiazolidinethione; BP, benzophenone; PA, phenylazide; MI, maleimide.
9 10 11
Cell permeability and localization of MRMs.
12
The cell permeability and mitochondrial localization of a set of MRMs in HeLa cells
13
were evaluated initially by confocal laser scanning microscopy (CLSM). Rapid (within 15
14
min) and spontaneous localization in mitochondria were observed for the majority of these
15
molecules. For example, the CLSM image of HeLa cells treated with 1a(CA) for 15 min is
16
shown in Figure 2a. This merged well with the image stained by Rhodamine123, a 7 ACS Paragon Plus Environment
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representative mitochondrial localization marker.20 Other MRMs such as 1b(EP), 1c(SF),
2
1d(TE), 1e(TZ), 1f(BP) and 1g(PA) gave almost identical CLSM images to the one observed
3
with 1a(CA) (Supplementary Figure S1). The excellent overlap and the resultant high
4
Pearson’s correlation coefficient (more than 0.85) indicated that these MRMs efficiently
5
accumulated in mitochondria of live HeLa cells. Such mitochondrial localization did not occur
6
in HeLa cells pretreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a standard
7
uncoupler of the mitochondrial membrane potential21 (Figure S2), revealing that spontaneous
8
accumulation of these MRMs was driven by the high membrane potential of mitochondria.
9
We also spectroscopically determined the intracellular concentration of these MRMs
10
after lysis of the HeLa cells (Figure S3). In the optimized conditions for the below-mentioned
11
protein labeling experiments, the concentrations of 1a(CA)–1g(PA) are in the range of 24 to
12
213 µM inside the cell. These values correspond to a 4 to 21-fold condensation relative to the
13
concentration of the MRMs added extracellularly. Given the volume of mitochondria in live
14
HeLa cells, it is regarded that the condensation effect is further enhanced by ~3-fold,22 which
15
could facilitate the intermolecular labeling reactions in mitochondria. In contrast, the
16
maleimide-appended Et4-Rhod 1h(MI) gave the rather weak intracellular fluorescence,
17
indicating its poor cell permeability (Figure S4). Hence, 1h was omitted from our set of
18
MRMs.
19 20
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(a)
(b) 1a(CA)
(kDa) 250 150
Rho 123
Fluorescence
CBB
100 75 50
Merge
Phase
37
25 Reagent 1a! 1b! 1c! 1d! 1e! 1f! 1g!
R = 0.900
2 3 4 5 6 7 8 9 10 11 12
20 µm
(CA) (EP) (SF) (TE) (TZ) (BP) (PA)
Conc.(µM) 10 10 10 Time (h) 6 6 6
5 2
5 5 10 2 0.5 0.5
Figure 2. Mitochondrial proteome labeling in HeLa cells with MRMs. (a) Confocal micrograph images of HeLa cells stained with 1a(CA) (1.0 µM) and Rhodamine 123 (200 nM). Colocalization was quantified using Pearson’s correlation coefficient (R). (b) SDS-PAGE analysis by in-gel fluorescence imaging and Coomassie Brilliant Blue (CBB) staining. Reaction conditions: HeLa cells were incubated with MRMs (5.0–10 µM) in serum-free DMEM at 37 °C for 0.5–6 h. In the case of 1f(BP) and 1g(PA), the cells were then UV irradiated (40 min) on ice. After labeling, the cells were washed, lysed and analyzed by SDS-PAGE. The optimization of labeling conditions with each MRM in living cells was shown in Figure S5 and S6.
13 14
Labeling and identification of proteins in mitochondria of HeLa cells.
15
Having a set of cell permeable MRMs in hand, we then evaluated their reactivity with
16
natural proteins in HeLa cells by SDS-PAGE analysis. After the addition of each MRM
17
(1a(CA)–1g(PA)) to the culture medium of HeLa cells, followed by incubation, the cells were
18
lysed and the resultant lysate was subjected to SDS-PAGE. As shown in Figure 2b, the
19
labeled protein bands were detected by fluorescence due to the Et4-Rhod fluorophore, which
20
indicated that the band pattern was distinct and dependent on the labeling reagent. In contrast
21
to the many bands observed for most MRMs, 1f(BP) gave only a few bands with noticeable
22
band intensity. In addition, the CLSM image after 1a(CA) treatment was not altered by the 9 ACS Paragon Plus Environment
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addition of CCCP, whereas clear mitochondrial images were not observed in the case of the
2
CCCP-pretreated HeLa cells (Figures S2 and S7). These data additionally support the
3
covalent attachment of 1a(CA) to proteins in mitochondria.
4
Identification of the labeled proteins in detail was subsequently carried out by
5
LC-MS/MS analysis. The Et4-Rhod fluorophore serves as a vehicle for targeting organelles
6
and is also an excellent antigen to elicit antibodies that should enrich labeled proteins and
7
peptides in downstream immunoprecipitation (IP) assays. We thus prepared an anti-Et4-Rhod
8
antibody23 and used this antibody for concentrating the labeled proteins/peptides prior to
9
MS/MS analysis. To directly detect the labeled peptides and to minimize the contamination
10
during IP and gel separation, we exploited the modified “gel-free” method that was originally
11
reported by Cravatt et al. (Figure 3a).24 In our protocol, the labeled peptides rather than
12
proteins were concentrated by IP, which were then analyzed by LC-MS/MS without
13
SDS-PAGE. In the case of 1a(CA), for example, we detected 373 labeled peptides, thus the
14
labeled amino acids were identified (Figure 3b, Table S1 and Supplementary data S1).
15
Clearly, Cys-labeled peptides (290 (78%)) were readily detected as the largest group. The
16
second largest groups were Ser- (8%) and Thr- (6%) labeled peptides, where the amino acid
17
preference is consistent with the intrinsic chemical reactivity of CA. A similar Cys preference
18
was also observed by 1b(EP) with labeling of 83% of the peptides and Ser-labeled peptides
19
were the second largest group (6%). On the other hand, 1d(TE) gave 82% of Lys-labeled
20
peptides and Ser-labeled peptides (12%) formed the second largest group. This preference was
21
almost the same as that of 1e(TZ) (90% for Lys). In the case of 1c(SF), it was characteristic
22
that Lys (46%) and Tyr (40%) were the two predominant amino acids labeled. Peptides
23
labeled by 1f(BP) and 1g(PA) were not detected, which may be due to the instability of the 10 ACS Paragon Plus Environment
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formed covalent bond and/or the formation of unexpected adducts. Of note, identifying
2
labeled peptides (not proteins) that we conducted here enabled us to address the amino acid
3
preference of these reactive moieties in live cells explicitly, which revealed that the chemical
4
selectivity in live cells roughly reflected those expected in conventional organic reactions.
5
Additionally, this strongly indicated that most of the detected labeled peptides did not come
6
from artifacts of the present MS fingerprinting protocol.
7
The data derived from these labeled peptide sequences were used to identify the labeled
8
proteins. Figure 3c and Table S2 summarizes the identified proteins and their localization.
9
The use of 1a(CA) labeled 244 proteins, and 196 of these proteins are assigned as
10
mitochondrial proteins (80%). Similarly, 85 (among 131), 53 (among 74), 52 (among 65) and
11
25 (among 57) mitochondrial proteins were identified by 1b(EP), 1c(SF), 1d(TE) and 1e(TZ),
12
respectively. Figure 3d and Figure S9 show a representative protein, trifunctional enzyme
13
subunit alpha, mitochondrial (HADHA), identified by 15 labeled peptides, where 12 sites
14
were modified with 5 MRMs. Similarly, 18 sites of 60 kDa heat shock protein, mitochondrial
15
(HSPD1) and 7 sites of Serine hydroxymethyltransferase, mitochondrial (SHMT2) were
16
labeled with 5 and 4 MRMs, respectively (Figure 3e, 3f and Figure S9). These results clearly
17
showed that different labeling reagents reacted with distinct peptide sequences and/or amino
18
acids (despite partial overlap) and more importantly these labeled residues were mainly
19
exposed on the protein surface and not buried in the active pocket. Such surface labeling was
20
observed in most of identified proteins whose 3D-structures were available or postulated25
21
(Figure S9 and S10), suggesting the mitochondria-localized labeling was facilitated by simple
22
intermolecular reactions in limited spaces,26 and not by specific activity/affinity with proteins
23
(Figure S12). It is also evident that the number and sort of identified proteins were largely 11 ACS Paragon Plus Environment
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dependent on the reactive moiety of the MRMs. More importantly, each MRM can label
2
unique proteins, while some overlap with each other. That is, Figure 3g and Table S3 show
3
that 105 mitochondrial proteins were uniquely identified by 1a(CA), and 10 by 1b(EP), 18 by
4
1c(SF), 14 by 1d(TE), and 4 by 1e(TZ). These results clearly highlight the advantage of using
5
a variety of chemically reactive moieties to cover a wide range of proteomes in mitochondria.
6
The annotation analysis of the whole labeled proteins revealed that 69% are assigned to
7
mitochondria-localizing proteins, 6% of the proteins were not assigned (un-assigned) to a
8
location and 25% were assigned as non-mitochondrial proteins (Figure 3c). The identified
9
mitochondrial proteins were further classified into submitochondrial localizations, namely,
10
“matrix”, “inner membrane (IMM)”, “intermembrane space (IMS)” and “outer membrane
11
(OMM)” (Figure 4a). Among 259 mitochondrial proteins identified in our study, 182 proteins
12
can be annotated by submitochondrial localization on the basis of the Gene Ontology (GO)
13
terms. We found that 163 proteins (66+33+64) were assigned to the matrix or IMM but not
14
IMS or OMM localization, while 19 proteins were assigned to the IMS or OMM localization.
15
That is, our MRM mainly labeled proteins localized in matrix or IMM of mitochondria.
16
Moreover, it was revealed that the coverage of OMM proteins, defined by the percentage of
17
identified OMM proteins over the whole OMM proteins, is lower (13%) than that of matrix
18
(48%), IMM (40%) and IMS (39%) (Figure 4b and 4c). These results clearly show our
19
MRMs mainly react with proteins inside mitochondria, which is consistent with the assumed
20
submitochondrial localization of Et4-Rhod.27 Among the un-assigned proteins, there are some
21
proteins shuttling between mitochondria and ER (or other organelles), such as Reticulon-428
22
and Apolipoprotein C-III29, which may be re-assigned as mitochondria-localized ones. The
23
25% non-mitochondrial proteins detected offers some caution to this method providing false 12 ACS Paragon Plus Environment
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positive information on protein localization. This is plausibly due to side reactions with highly
2
abundant proteins such as cytoskeletal proteins30, the misdistribution of the MRMs during the
3
labeling process and mischaracterization of the labeled peptides during LC-MS/MS analysis.
4
However, as mentioned above, these MRMs were efficiently concentrated in mitochondria of
5
live HeLa cells. Additionally, negative control experiments using the HeLa lysate and
6
CCCP-pretreated HeLa cells as the labeling sample (1a(CA)) confirmed that the content of the
7
mitochondria-localizing proteins decreased to only 13 and 16%, respectively (Figure 4d,
8
Figure S13 and Supplementary data S5). Given these results, it is concluded that most of
9
MRMs mainly react with mitochondrial proteins by spontaneous condensation in the
10
mitochondrial limited space under live cell conditions and therefore MRMs are suitable tools
11
for mitochondria-selective chemical proteomics.
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(a)
Digestion
IP
LC-MS/MS m/z
Labeled Proteome
Crude Peptides
Labeled Peptides
(b)
m/z
(c) 400% 350%
S28%
300% 250% 200%
S13%
150%
C290%
100%
C166%
50%
S15%
Y46%
K100%
K53%
K70%
0% 1a(CA)% 1b(EP) 1b(EP)% 1a(CA)
1c(SF)% 1c(SF)
1d(TE)% 1d(TE)
69% 21$
400$
Y% T% S% K% H% E% D% C%
Number of iden-fied proteins
Number of identified peptide
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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350$
Mitochondrial
300$
80%
250$
38$
200$
10$
150$
196$
100$ 50$ 0$
1e(TZ)% 1e(TZ)
95$
Other Organellar
CA$ 1a(CA)
Un-assigned 65% 11$ 72% 35$ 21$ 85$ 53$ EP$ 1b(EP)
80% 9$
259$
44% 4$
52$
SF$ TE$ 1c(SF) 1d(TE)
28$
4$ 25$
TZ$ 1e(TZ)
Y343 (1c)
(d) K334 (1c)
H598 (1c)
C322 (1b) K570 (1d)
K309 (1d) T294 (1d)
(e)
active site
K569 (1c)
K295 (1d)
K646 (1c, 1d, 1e)
(f)
K292 (1d, 1e)
C80 (1a) C91 (1a)
K202 (1c, 1d)
T406 (1d)
K205 (1c, 1d, 1e ) K71 (1c, 1d)
active site
K82 (1c, 1d) K96 (1c, 1d, 1e ) H267 (1c) Y385 (1c)
K133 (1d)
Y106 (1c)
K157 (1d)
(g) Overlapped
Unique Identified 259 mitochondrial proteins
1a(CA) 1b(EP) 1c(SF) 1d(TE)
1
1e(TZ)
2 3 14 ACS Paragon Plus Environment
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Total$ Total
Page 15 of 36
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Figure 3. Identification and profiling of Et4-Rhod labeled proteins and their modified residues. (a) Workflow of labeled-peptide/protein identification by the gel-free protocol. (b) The number of identified peptides and their labeled residues determined by LC-MS/MS analysis. Y, tyrosine; T, threonine; S, serine; K, lysine; H, histidine; E, glutamic acid; D, aspartic acid; C, cysteine. (c) The number of proteins identified by LC-MS/MS analysis from HeLa cells and the ratio of mitochondrial proteins. The identified proteins were classified by cellular location using the protein ontology information provided in the UniProt database. (d, e, f) 3D structures of (d) HADHA, (e) HSPD1 and (f) SHMT2 with labeled residues (colored). These protein models from SWISS-MODEL were manipulated and rendered in PyMOL. Additional images of these proteins from different viewpoints and other representative proteins are showed in Figure S9 and S10. (g) Color map representation of identified mitochondrial proteins. Proteins that were uniquely or overlappingly identified by each MRM are highlighted in blue or red, respectively. Detailed information on identified proteins is presented in Supplementary data S1.
(a)
(b)
Unclassified 77
(d)
(c)
Mitochondrial
18 19 20 21 22 23 24 25
Submitochondrialspaces
Identified
Whole
Coverage
Matrix
103
215
48%
IMM
109
270
40%
IMS
12
31
39%
OMM
7
53
13%
Number of iden-fied proteins
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society
200"
13%
150" 100"
16% 154! 98!
50" 0"
Other organellar and un-assigned
24! lysate" lysate
18! CCCP" CCCP(+)
Figure 4. Submitochondrial localization of identified proteins and reaction specificity of MRMs. (a) Venn diagram representing the submitochondrial localization of identified proteins from HeLa cells. Based on the GO terms, proteins identified as mitochondrial proteins were further classified into “matrix”, “inner membrane (IMM)”, “intermembrane space (IMS)” and “outer membrane (OMM)”. It should be noted that some proteins are classified into plural submitochondrial regions. The list of identified proteins with their submitochondrial localization can be found in Supplementary data S1. (b) Venn diagram of submitochondrial 15 ACS Paragon Plus Environment
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1 2 3 4 5 6
Page 16 of 36
localization of whole mitochondrial proteins with prior submitochondrial localization data in MitoCarta31 or Uniprot database. (c) Coverage of identified proteins in each submitochondrial spaces. Identified, the number of identified proteins; Whole, the number of mitochondrial proteins registered in the databases. (d) The number of identified proteins and the ratio of mitochondrial proteins in negative control experiments. HeLa cell lysate and CCCP-pretreated HeLa cells were incubated with 1a(CA), followed by LC-MS/MS analysis.
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1
Journal of the American Chemical Society
Application of MRMs to primary cultured neurons and brain slices.
2
Our MRMs-based chemical proteomics method requires neither pre-treatment of cells
3
such as gene transfection, nor careful (and time-consuming) organelle fractionation processes.
4
Thus, this method can be applied to mitochondria-focused proteomics of primary cells and/or
5
more complicated natural samples such as brain tissue. As a representative example, we
6
explored the mitochondrial proteome of primary cultured neurons. Each of the five MRMs
7
were mixed with cultured neural cells isolated from the cerebral cortex of the rat brain,
8
followed by incubation. The localization of each MRM was confirmed by CLSM imaging
9
(Figures 5a, Figure S14 and Supplementary data S1), showing that the MRMs
10
spontaneously accumulated to mitochondria of the rat neuron. Using a similar protocol
11
employed for the HeLa cells, SDS-PAGE analysis and gel-free protein identification were
12
conducted (Figures 5b, 5c and Supplementary data S2). Interestingly, the amino acid
13
preference of each MRM determined by the labeled peptides was almost identical with the
14
experimental data obtained from HeLa cells (Table S4). Additionally, we identified 212
15
proteins that included at least 129 (~61%) mitochondrial proteins using 5 MRMs (Figure 5c
16
and Table S5).
17
Moreover, the mitochondrial proteomics of brain slices was accomplished by mixing
18
each MRM with acutely prepared brain slices of mice cerebral cortexes. As shown in Figure
19
5d, labeled protein bands were observed in all MRMs, while the band intensity of 1b(EP) was
20
quite weak when compared with the labeling in primary neurons. This is probably due to
21
1b(EP) poor tissue permeability. One hundred proteins were assigned as mitochondrial
22
proteins from this brain slice experiments, which corresponded to 64% of the total number of
23
proteins identified (Figure S15, Table S6 and Supplementary data S3). This ratio and amino 17 ACS Paragon Plus Environment
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1
acid preference are in agreement with those of HeLa cells and primary cultured neurons
2
(Table S7). We subsequently classified these proteins on the basis of their function using
3
DAVID bioinformatics resources (Figure 5e).32 It was revealed that the majority of the
4
proteins labeled in the brain slices are involved with unique mitochondrial functions, such as
5
oxoacid metabolic process, electron transport chain and energy derivation by oxidation of
6
organic compounds. This labeling pattern is functionally identical with that obtained in
7
labeling the primary neurons with the MRMs. Although the coverage of mitochondrial
8
proteins are not sufficiently high likely due to the low tissue-permeability of MRMs, our
9
MRM-based method clearly has valuable features: (i) simplicity of the experimental protocol;
10
(ii) minimization of unfavourable effects to live samples; and (iii) successful chemical
11
proteomics of mitochondria for sensitive and complicated biological samples that are not
12
easily amenable to exogenous gene manipulation.8
13 14
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(a)
(c)
(b) 1a(CA)
Fluorescence
(kDa) 250 150
Rho 123
250#
Number'of'iden-fied'proteins
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society
100 75 50
Merge
Phase 37
200#
Other Organellar 150#
63% 11#
100#
39# 63%
50#
84#
13# 25#
0#
Reagent 1a! 1b! 1c! 1d! 1e! (CA) (EP) (SF) (TE) (TZ)
Conc.(µM) 1.0 5 Time (h) 6 6
(d) (kDa)
72#
Un-assigned
25 20 µm
61% 11#
Mitochondrial
CA# 1a! (CA)
5 2.5 2.5 6 2 2
83%
2# 7#
EP# 1b! (EP)
68% 15#
69% 18#
34#
32#
40#
SF# 1c! (SF)
TE# 1d! (TE)
TZ# 1e! (TZ)
129#
Total# Total
(e)
Fluorescence
250 150
oxoacid"metabolic"process" oxoacid metabolic process!
100 electron"transport"chain" electron transport chain
75 50
Cultured Neurons Brain Slices
energy"deriva9on"by"oxida9on"of"organic" energy derivation by oxidation ! compounds" of organic compounds!
37
cofactor"catabolic"process" cofactor catabolic process! coenzyme"metabolic"process" coenzyme metabolic process!
Reagent 1a! 1b! 1c! 1d! 1e!
0" 0
(CA) (EP) (SF) (TE) (TZ)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
5" 5
10" 10
15" 15
2020" (%)
Conc.(µM) 5.0 15 15 15 15 Time (h) 6 6 6 2 2
Figure 5. Mitochondrial proteome labeling in primary cortical neurons and brain slices with MRMs. (a) Confocal micrograph images of neurons stained with 1a(CA) (1.0 µM), Rhodamine 123 (200 nM) and Hoechst 33342 (1.0 µM). (b) SDS-PAGE analysis by in-gel fluorescence imaging. Reaction conditions: primary cortical neurons 6 × 105 cells (16-d-old Sprague Dawley rat embryos) were incubated with MRMs (1.0–5.0 µM) in serum-free DMEM at 37 °C for 2–6 h. After labeling, the cells were washed, lysed and analyzed by SDS-PAGE. (c) The number of proteins identified by LC-MS/MS analysis from primary cultured neurons and the ratio of mitochondrial proteins. (d) SDS-PAGE analysis by in-gel fluorescence imaging. Reaction conditions: Brain (cerebrum) slices (250 µm thickness) prepared from P14–21 ICR mice were incubated with MRMs (5.0–15 µM) in the ACSF solution at room temperature for 2–6 h under 95% O2 / 5% CO2. After labeling, the tissues were washed, lysed and analyzed by SDS-PAGE. (e) Functional classification of identified proteins from primary cultured neurons and brain slices by LC-MS/MS analysis (enrichment P-value by DAVID < 0.0002).
18 19
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Chemical profiling of a dynamic proteome during an oxidant-induced apoptosis process
2
We finally applied this MRM approach to explore the dynamics of mitochondrial
3
proteome of live cells in response to a cellular stress. As a proof-of-principle, we combined
4
our MRM-based method with SILAC, a quantitative MS analysis technique,33 in order to
5
investigate the change of mitochondrial proteome of HeLa cells during an apoptosis process
6
induced by paraquat (PQ),34 an oxidative stress inducer. HeLa cells grown in light SILAC
7
media were incubated with PQ for 48 h, followed by 1a(CA) (5.0 µM, 6 h), whereas cells
8
grown in heavy SILAC media were only treated with 1a(CA) in the absence of PQ (Figure
9
6a). The PQ-induced apoptosis was validated by a cleavage of PARP-1, a standard marker of
10
apoptosis (Figure S16). It was also confirmed that 1a(CA) localized in mitochondria of the
11
apoptotic HeLa cells like as normal cells (Figure S17) and the intracellular concentration did
12
not substantially change by PQ treatment (Figure S18). As shown in Figure S19, the
13
fluorescent band patterns of SDS-PAGE gel were well identical between the PQ-treated and
14
PQ-nontreated cells, indicating the PQ-treatment did not largely impact most of mitochondrial
15
proteins of HeLa cells. To obtain the optimal performance for quantitative MS analysis, we
16
carried out a standard protein identification protocol including IP of the labeled proteins (not
17
peptides), followed by MS fingerprinting analysis. 255 proteins were identified and quantified
18
in total from three independent biological replicates (Figure S20), among which 166 proteins
19
(65%) were classified into mitochondrial proteins, showing that our MRM-based labeling
20
works well in apoptotic cells (Figure 6b and Supplementary data S4). Of the quantified
21
proteins, 21 proteins were up-regulated by more than 2-fold in PQ-treated HeLa cells (Table
22
S8), which included “glutathione peroxidase 1 (GPX1)”, “Peroxiredoxin-1 (PRDX1)” and
23
“Peroxiredoxin-2
(PRDX2)” that have been reported to increase under oxidative stress or 20 ACS Paragon Plus Environment
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Journal of the American Chemical Society
1
apoptotic processes.35 Functional annotation analysis of the 21 up-regulated proteins using
2
DAVID revealed that terms of apoptosis-related biological process mediated by reactive
3
oxygen species (ROS), such as “regulation of apoptosis” (36%, p value: 1.54 × 10-3),
4
“regulation of programmed cell death”
5
peroxide” (18%, p value: 6.41 × 10-3), “cellular response to reactive oxygen species” (18%, p
6
value: 1.21 × 10-2), and “positive regulation of programmed cell death” (23%, p value: 1.25 ×
7
10-2), were significantly enriched (Figure S21). It was also found that 7 mitochondrial
8
proteins were down-regulated by more than 2-fold (Table S9), among which 3 proteins
9
(NDUFS3, NDUFS1 and FASN) are previously reported to decrease under oxidative stress or
10
apoptotic processes.36 These data obtained by MRM-based SILAC technique consistently
11
reflect the dynamic change of mitochondrial proteome of HeLa cells in response to the
12
oxidative stress.
(36%, p value: 1.54 x 10-3), “response to hydrogen
13
In order to further evaluate our quantitative MS data, western blotting (WB) analysis
14
was carried out. We selected 6 representative mitochondrial proteins from the list of identified
15
proteins with substantial (>2-fold) changes in the SILAC experiment. These include three
16
up-regulated proteins (AIFM1, ACO2, GPX1) and three down-regulated proteins (NDUFAF4,
17
NDUFS3, FASN). Among them, three proteins (AIFM1, ACO2, NDUFAF4) are previously
18
unknown to change in the expression levels through apoptosis, whereas substantial change of
19
the expression level for the other three (GPX1, NDUFS3, FASN) are already reported.
20
Besides, two proteins (HADHA, HSPA9) showing not substantial alterations in the SILAC
21
ratios were also evaluated by WB analysis. As shown in Figure 6c, WB data for seven
22
proteins, AIFM1, GPX1, NDUFAF4, NDUFS3, FASN, HADHA and HSPA9 indicated the
23
same tendencies in the change of the expression levels as that determined by the SILAC 21 ACS Paragon Plus Environment
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Page 22 of 36
1
experiment. These results clearly validated the data of our quantitative MS analysis exploiting
2
MRM-based labeling strategy. Notably, both up-regulation of AIFM1 and down-regulation of
3
NDUFAF4 in the ROS-mediated apoptosis process are identified for the first time, to the best
4
of our knowledge. AIFM1, apoptosis-inducing factor 1, is an NADH oxidase and implicated
5
in a caspase-independent pathway of programed cell death.37 NDUFAF4, NADH
6
dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 4 which is involved in the
7
assembly of respiratory chain complex I, is reported to negatively regulates apoptosis.38
8
Importantly, these proteins are supposed to be associated with some of mitochondrial
9
diseases,39 such as neuropathy and Parkinson disease. Thus, the present new finding may
10
encourage us to facilitate a more detailed study on biological roles of these proteins in
11
dysfunctional mitochondria.
12
On the other hand, WB data of ACO2, mitochondrial aconitate hydratase, did not
13
agree with that of the SILAC experiment. To examine this discrepancy, we focused on the
14
labeling sites of ACO2. MS analysis successfully identified Cys385 and Cys451 as the
15
labeling sites, both of which are involved in binding to an iron-sulfur [4Fe-4S] cluster, an
16
active site of ACO2. It is also reported that this [4Fe-4S] cluster is destructed and removed by
17
ROS,40 leading to a conformational change of ACO2 and its subsequent degradation. Given
18
these reports, it is reasonably considered that the removal of [4Fe-4S] cluster from ACO2 by
19
PQ-induced ROS enhances the accessibility (i.e. reactivity) of the Cys385 and Cys451 with
20
MRMs, resulting in an increase of the SILAC ratio despite of a decline of ACO2 expression
21
level. While this example may point out a false positive case of our MRM-based SILAC
22
approach, it also suggests that the careful examination of our data could give us interesting
23
information on the structure and/or activities changes of a mitochondrial protein in living 22 ACS Paragon Plus Environment
Page 23 of 36
1
cells.
2
Overall, these experiments undoubtedly demonstrate that our MRM-based method
3
provides a convenient and quantitative approach to efficiently focus on representative proteins
4
in the dynamic change of mitochondrial proteins, in combination with a standard quantitative
5
proteomic technique.
6 7 (a)
PQ
PQ-treated SILAC “light”
48 h
labeling
Mix lysate" (1 : 1)
IP"
in-gel" digestion
SDS-PAGE
LC-MS/MS
vehicle
Control SILAC “heavy”
(b)
!mitochondrial
PQ
AIFM1!
Up-regulated " (> 2-fold)
2!
(c)
Other Organellar" and un-assigned"
3!
log2 PQ / Control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society
AIFM1 ACO2"
1!
GPX1 HADHA!
HADHA
0!
HSPA9 HSP9A!
-1!
FASN
FASN!
Down-regulated " (> 2-fold)
NDUFAF4
-3! 0!
50!
WB SILAC
NDUFS3
NDUFS3! NDUFAF4!
9 10 11 12 13 14 15 16 17 18 19 20 21 22
+
ACO2! GPX1!
-2!
8
–
100!
150!
200!
-3! -3
250!
-2! -2
-1! -1
0! 0
1! 1
2! 2
33!
log2 (PQ/Control)
Protein
Figure 6. Quantitative MS analysis of the change of mitochondrial proteome under an oxidative stress. (a) Schematic illustration of SILAC experiments coupled with MRMs to quantify mitochondrial proteome changes in paraquat (PQ)-treated HeLa cells. PQ-treated cells grown in the light SILAC medium and non-treated cells grown in the heavy SILAC medium (control) were labeled with 1a (CA) and lysed. Protein extracts from the light and heavy cells were mixed in a 1:1 ratio, and then the Et4-Rhod-labeled proteins were enriched by IP using anti-Et4-Rhod antibody, followed by in-gel tryptic digestion and LC-MS/MS analysis. (b) Log2 ratio plot (PQ-treated (light) cells/control (heavy)) of identified proteins. Proteins assigned as mitochondrial are colored in red in the plot. Proteins without mitochondrial annotation are colored in black. (c) Western blotting analysis of PQ-induced changes in expression levels of the selected proteins (n=3 (±standard deviation)) and their comparision with data from SILAC.
23 23 ACS Paragon Plus Environment
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Conclusions
2
We have developed a set of MRMs as novel chemical reagents for organelle-focused
3
proteomics. This gave us a remarkably simple experimental protocol of organelle-selective
4
labeling that is compatible with conventional LC-MS/MS and MS-fingerprinting techniques.
5
This method explicitly addressed the chemical reactivity and selectivity of various reactive
6
groups in live cells and identified the mitochondrial proteome in essentially natural biological
7
habitats, including live primary cultured neurons, brain slices and HeLa cells. Quantitative
8
profiling and characterizing particular proteins have also been accomplished using this method,
9
whose expression levels were found to change dynamically during an oxidant-induced
10
apoptosis.
11
The present MRM-based method was able to label and identify a relatively small
12
fraction of the mitochondrial whole proteome that is roughly estimated to contain 1,000–1,500
13
proteins.4, 41 The limited coverage may be partially attributed to the biased submitochondrial
14
distribution of the Et4-Rhod-based MRMs (in the matrix and inner membrane). This issue
15
would be overcome by varying the localizable moiety to target different areas within the
16
mitochondria.42 Moreover, given the modular structures of MRMs, a rational extension of this
17
strategy targets other organelles by varying the localizable moiety to cover a whole proteome
18
in live cells and tissues. We envision that the sets of organelle-localizable reactive molecules
19
can be widely used against a variety of intact cells involved in organelle-relevant diseases to
20
identify molecular details by profiling differentiating proteomes at high resolution.
21 22 23 24 ACS Paragon Plus Environment
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Journal of the American Chemical Society
1
Experimental Section
2
Synthesis.
3 4
All synthetic procedures and compound characterizations are described in the Supporting Information.
5 6
General materials and methods for the biochemical/biological experiments.
7
Unless otherwise noted, all proteins/enzymes and reagents were obtained from commercial
8
suppliers (Sigma-Aldrich, Tokyo Chemical Industry (TCI), Wako Pure Chemical Industries,
9
Sasaki Chemical, Bio-Rad, or Watanabe Chemical Industries) and used without further
10
purification.
UV-vis
absorption
spectra
were
acquired
on
a
Shimazu
UV-2550
11
spectrophotometer. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western
12
blotting were carried out using a Bio-Rad Mini-Protean III electrophoresis apparatus.
13
Fluorescence gel images and chemical luminescent signals using Chemi-Lumi one (Nacalai
14
Tesque) or ECL Prime (GE Healthcare) were acquired with an imagequant LAS4000 (GE
15
Healthcare). Cell imaging was performed with a confocal laser scanning microscope (CLSM,
16
Olympus, FV10i) using 60× objectives, and images were analyzed using accompanying
17
FV10i-ASW 3.0 Viewer software.
18 19
CLSM analysis of HeLa cells
20
HeLa cells (2.0 × 105 cells) were cultured on 35 mm glass based dishes in Dulbecco’s
21
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
22
Antibiotic-Antimycotic (Anti-Anti, gibco) under a humidified atmosphere of 5% CO2 in air at
23
37 °C for 24 h. The cells were washed twice with serum-free DMEM (HEPES modified, no
24
Phenol Red), and incubated in serum-free DMEM containing Et4-Rhod-type MRM (1.0 µM)
25
for 15 min or Hoechst-type MRM (10 µM) for 60 min. The intracellular localization of these
26
reagents was analyzed by CLSM. As a negative control for mitochondrial-localizable reagents,
27
HeLa cells were preincubated with CCCP (5.0 µM) for 5 min before addition of
28
Et4-Rhod-type MRMs.
29 30
Mitochondrial proteome labeling with Et4-Rhod-type MRMs in HeLa cells 25 ACS Paragon Plus Environment
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1
HeLa cells (1.0 × 106 cells) were cultured on 10 cm dishes under a humidified atmosphere of
2
5% CO2 in air at 37 °C for 48 h in DMEM. The cells were washed twice with serum-free
3
DMEM, and incubated in serum free DMEM containing Et4-Rhod-type MRM (5–10 µM) for
4
0.5–6 h. In the case of using photoreactive probes, such as 1f(BP) and 1g(PA), the cells were
5
washed twice with chilled PBS, and then exposed to 365 nm light with a handy UV lamp (4
6
W) at 1 cm distance from the top of cell culture dish for 40 min on ice. After labeling reaction,
7
the cells were washed twice with chilled PBS, lysed by sonication in modified-RIPA buffer
8
(25 mM Tris, 150 mM NaCl, 1% Sodium dodecyl sulfate, 1% Nonidet P-40, 1% sodium
9
deoxycholate, pH 7.6) containing 1% protease inhibitor cocktail set III (Novagen) at 4 °C.
10
Proteins were precipitated with chilled acetone, and the pellets were solubilized by sonication
11
in modified-RIPA buffer. The solubilized proteins were normalized for protein concentration
12
(BCA assay kit, Thermo) to 1.0 mg/mL, and then mixed with 1/4 volume of 5 × SDS–PAGE
13
loading buffer (pH 6.8, 325 mM Tris, 20% sucrose, 15% SDS, 0.03% bromophenol blue)
14
containing 250 mM DTT and vortexed for 30 min. The samples were resolved by SDS-PAGE
15
(8.0 µg protein/lane), and the Et4-Rhod labeled proteins were visualized fluorescence gel
16
imager (LAS4000, GE Healthcare).
17 18
Preparation of LC-MS samples (in-solution digested samples).
19
After mitochondrial proteome labeling with Et4-Rhod-type MRMs, the cells were lysed with
20
RIPA buffer (25 mM Tris, 150 mM NaCl, 0.1% Sodium dodecyl sulfate, 1% Nonidet P-40,
21
1% sodium deoxycholate, pH 7.6). All proteins were precipitated with chilled acetone, and
22
solubilized by sonication in modified-RIPA buffer. Protein concentration was determined by
23
BCA assay and normalized to 2.0 mg/mL (total volume 1 mL). The samples were diluted into
24
1 mL of 12 M urea (final conc. 6 M), reduced with 20 mM DTT at 65 °C for 5 min, and
25
alkylated with 40 mM iodoacetamide at 37 °C for 30 min. After precipitation with 20%
26
methanol containing acetone (8 mL), 500 µg of proteins were treated with following two
27
methods (method A or B).
28
In method A, the pellet of proteins was solubilized by sonication in 500 µL of sodium
29
deoxycholate (SDC) buffer (0.1% SDC, 1M urea, 10 mM ammonium bicarbonate), and
30
digested with 5 µg of trypsin (Thermo, MS Grade) overnight at 37 °C. The mixture was boiled 26 ACS Paragon Plus Environment
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Journal of the American Chemical Society
1
at 65 °C for 5 min to inactivate trypsin. To remove the surfactant, phase-transfer surfactant
2
protocols were conducted.24 Briefly, the digested solution was mixed with 500 µL of ethyl
3
acetate, acidified with 2.5 µL of TFA, and shaken for 1 min. The mixture was centrifuged at
4
16000 × g for 2 min, and the organic phase was removed. The aqueous phase was washed with
5
500 µL of ethyl acetate again, and neutralized with 200 mM Tris-HCl (pH 7.4).
6
In method B, 500 µg of proteins were solubilized by sonication in 250 µL of urea buffer (4M
7
urea, 100 mM Tris-HCl, pH 8.0). The solution was diluted with 250 µL of 100 mM Tris-HCl
8
(pH 8.0), and incubated with 5 µg of trypsin overnight at 37 °C. The mixture was boiled at
9
65 °C for 5 min and diluted with 1.5 mL of Tris-HCl (pH 8.0).
10
The resulting solutions in method A and B were incubated with 5.0 µL of anti-Et4-Rhod
11
antiserum for 6 h at 4 °C with continuous rotation, followed by addition of 50 µL of nProtein
12
A sepharose 4 Fast Flow (GE Healthcare, 50% slurry in PBS) and further rotation at 4 °C for 2
13
h. The beads were washed 5 times with PBS (1 mL) and water (1 mL). The peptides labeled
14
with Et4-Rhod were eluted with 40 µL of 50% water/CH3CN (0.1% TFA) × 2 and 40 µL of
15
CH3CN (0.1% TFA) × 2.These eluents were collected and centrifuged (12000 × g, 5 min). The
16
supernatant was freeze dried, redissolved with 20 µL of 2% CH3CN/water containing 0.1%
17
TFA, and further centrifuged at 16000 × g for 5 min. This supernatant (2–4 µL) was subjected
18
to LC-MS analysis. The list of identified proteins obtained by method A and B were integrated
19
into an output file. Protein identification experiments using HeLa cells and primary cortical
20
neuronal culture were independently performed twice.
21 22
Preparation of LC-MS samples (SILAC samples)
23
HeLa cells were grown in “heavy” SILAC media (DMEM, Thermo) containing 100 µg/mL of
24
13
25
Laboratories)
26
14
27
humidified atmosphere of 5% CO2 in air at 37 °C. The cells were passaged at least five times
28
in the SILAC media before being used for analysis. The isotope-labeled cells (6.0 × 105 cells)
29
were seeded on 10 cm dishes and incubated for 24 h in the SILAC media. The “light” labeled
30
cells were incubated with light SILAC media containing Paraquat (PQ) (100 µM) for 48 h,
C6 ,
15
N2-Lys (Cambridge Isotope Laboratories) and or “light” SILAC media containing
13 12
C6 ,
C6 ,
15 14
N4,-Arg (Cambridge Isotope
N2-Lys (Thermo) and
12
C6 ,
N4,-Arg (Thermo) supplemented with 10% dialyzed FBS (Gibco) and 1% Anti-Anti under a
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Page 28 of 36
1
while the “heavy” labeled cells were treated with heavy SILAC media containing vehicle
2
(water). Both the light and heavy cells were washed twice with PBS, and incubated in serum
3
free SILAC media containing 1a(CA) (5.0 µM) for 6 h. After labeling reaction, the cells were
4
washed twice with chilled PBS, lysed by sonication in modified-RIPA buffer containing 1%
5
protease inhibitor cocktail at 4 °C. Proteins were precipitated with chilled acetone, and the
6
pellets were solubilized by sonication in modified-RIPA buffer. The solubilized proteins were
7
normalized for protein concentration with BCA assay. Equal amounts of protein from heavy
8
(500 µg/250 µL) and light (500 µg/250 µL) samples were mixed in a 1 : 1 ratio, and the
9
mixture were diluted with PBS into 1.0 mL. The samples were precleared against 100 µL of
10
nProtein A sepharose 4 Fast flow at 4 °C for 30 min. After centrifugation, the supernatant was
11
collected and incubated with anti-Et4-Rhod (10.0 µL) at 4 °C overnight with continuous
12
rotation, followed by addition of 100 µL of nProtein A sepharose 4 Fast flow. After incubation
13
at 4 °C for 2 h, the beads were washed 5 times with RIPA buffer (1.0 mL), and then boiled in
14
2× SDS-PAGE sample buffer (25 µL) containing 100 mM DTT for 3 min. The supernatant
15
(15 µL) was loaded to SDS-PAGE. The samples were resolved 1.5 cm by the running gel and
16
fixed with 45% methanol/water containing 5% acetic acid for 20 min. The fixed gel was
17
washed with 50% methanol/water and water, and manually cut into 8 gel slices without gel
18
staining. The excised gels were subjected to in-gel digestion using trypsin (In-Gel Tryptic
19
Digestion Kit, Thermo Fisher Scientific, trypsin 200 ng/slice). The eluted peptide solution
20
from the gel was freeze dried and redissolved with 18 µL of 2% CH3CN/water containing
21
0.1% TFA. The solutions (4.0 µL) were subjected to LC-MS analyses. Three independent
22
SILAC experiments were performed, in which proteins detected at least twice with paired
23
light- and heavy-labeled peptides were included in the datasets as identified proteins.
24 25
LC-MS analysis
26
After proteome labeling, proteins were digested with trypsin in solution or in gel. The samples
27
for protein identification were analyzed by nano-flow reverse phase liquid chromatography
28
followed by tandem MS, using a LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher
29
Scientific). For LTQ-Orbitrap XL, a capillary reverse phase HPLC-MS/MS system composed
30
of an Agilent 1100 series gradient pump equipped with Valco C2 valves with 150-µm ports, 28 ACS Paragon Plus Environment
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Journal of the American Chemical Society
1
and LTQ-Orbitrap XL hybrid mass spectrometer equipped with an XYZ nano-electrospray
2
ionization (NSI) source (AMR). Samples were automatically injected using PAL system (CTC
3
analytics, Zwingen, Switzerland) into a peptide L-trap column OSD (5 µm, AMR) attached to
4
an injector valve for desalinating and concentrating peptides. After washing the trap with
5
MS-grade water containing 0.1% trifluoroacetic acid and 2% acetonitrile (solvent C), the
6
peptides were loaded into a nano HPLC capillary column (C18-packed with the gel particle
7
size of 3 µm, 0.1 × 150 mm, Nikkyo Technos) by switching the valve. The eluents used were:
8
A, 100% water containing 0.1% formic acid, and B, 80% acetonitrile containing 0.1% formic
9
acid. The column was developed at a flow rate of 200–500 nl/min with the concentration
10
gradient of acetonitrile. Xcalibur 2.1 system (Thermo Fisher Scientific) was used to record
11
peptide spectra over the mass range of m/z 350–1500, and MS/MS spectra in
12
information-dependent data acquisition over the mass range of m/z 150–2000. Repeatedly,
13
MS spectra were recorded followed by three data-dependent collision induced dissociation
14
(CID) MS/MS spectra generated from three or eight highest intensity precursor ions for the
15
non-quantitative MS or SILAC experiment, respectively. Multiple charged peptides were
16
chosen for MS/MS experiments due to their good fragmentation characteristics. MS/MS
17
spectra were interpreted and peak lists were generated by Proteome Discoverer 1.4 (Thermo
18
Fisher Scientific). Searches were performed by using the SEQUEST-HT (Thermo Fisher
19
Scientific)
20
carbamidomethylation on cysteine and oxidation on methionine were searched with peptide
21
mass tolerance at 10 or 20 ppm for non-quantitative MS or SILAC experiment, respectively;
22
MS/MS mass tolerance of 0.6 Da. Multiple Et4-Rhod modified peptides were omitted from
23
the datasets. The resultant data set was filtered to a maximum false discovery rate (FDR) of
24
0.01. Identified peptides with same sequence were counted as the identical peptides.
against
uniprot
database.
Dynamic
modifications
of
Et4-Rhod,
25 26 27
Acknowledgements
28
We thank Nozomi Ogawa, Karin Nishimura and Youhei Tatsukami (Kyoto University) for
29
experimental support of LC-MS and helpful discussion. We also thank Sho Wakayama (Kyoto
30
University) for preparation of brain slices. Y.Y. (26-4745) is supported by Japan Society for 29 ACS Paragon Plus Environment
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Page 30 of 36
1
the Promotion of Science (JSPS) for doctoral fellowships. This work was funded by the Japan
2
Science and Technology Agency (JST) Core Research for Evolutional Science and
3
Technology (CREST) and by JSPS KAKENHI (15H01637) to I.H.
4 5
Competing financial interests
6
The authors declare no competing financial interests.
7 8 9
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