Chemical Profiling of the Endoplasmic Reticulum Proteome Using Designer Labeling Reagents

Article abstract of DOI:10.1021/jacs.8b08606

The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem mass spectrometry (LC-MS/MS). Using this method, we performed proteomic analysis of the ER of HeLa cells and newly assigned three proteins, namely, PAICS, TXNL1, and PPIA, as ER-associated proteins. The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.

Full text of DOI:10.1021/jacs.8b08606

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Article
Chemical profiling of the endoplasmic reticulum
proteome using designer labeling reagents
Alma Fujisawa, Tomonori Tamura, Yuki Yasueda, Keiko Kuwata, and Itaru Hamachi
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08606 • Publication Date (Web): 15 Nov 2018
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Chemical profiling of the endoplasmic reticulum
proteome using designer labeling reagents
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Alma Fujisawa1, Tomonori Tamura1, Yuki Yasueda1, Keiko Kuwata2, Itaru Hamachi1,3,*
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
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2Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Chikusa, Nagoya 464-8602,
Japan
3Core Research for Evolutional Science and Technology, Japan Science and Technology Agency,
Sanbancho, Chiyodaku, Tokyo 102-0075, Japan
*Correspondence should be addressed to I.H. (ihamachi@sbchem.kyoto-u.ac.jp).
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Abstract
1
The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions
via interactions with other organelles. Despite its important role, chemical tools for profiling the
composition and dynamics of ER proteins remain very limited because of the labile nature of these
proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for
ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and
selectively label ER-associated proteins with a combined affinity and imaging tag, enabling
tag-mediated ER protein enrichment and identification with liquid chromatography tandem-mass
spectrometry (LC-MS/MS). Using this method, we performed proteomic analysis of the ER of HeLa
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cells and newly assigned three proteins, namely PAICS, TXNL1 and PPIA, as ER-associated proteins.
The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable
reactive molecules previously developed by our group, which enabled orthogonal organellar
chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic
changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by
combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell
culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical
proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.
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Introduction
1
The endoplasmic reticulum (ER) is an essential subcellular component responsible for the
synthesis, processing, and transport of proteins.1 Virtually all plasma membrane-associated proteins
and secretory proteins are synthesized in the ER and delivered to their final destination through
vesicular trafficking.2 It is increasingly recognized that communication between the ER and other
organelles, such as the mitochondria, Golgi apparatus, and endosomes, plays a pivotal role in diverse
cellular functions.3,4 In addition, recent reports suggest that malfunctions of the ER induced by
cellular stress is implicated in a variety of diseases, including diabetes, inflammation, and
neurodegenerative disorders.5 Thus, comprehensive characterization of ER-associated proteins
should be useful not only as a fundamental biological study but also for understanding the etiology of
such diseases. Conventional proteomic studies of the ER have mainly relied on subcellular
fractionation with sucrose gradient ultracentrifugation, followed by mass spectrometry (MS) or
gel-based analysis.6–8 However, isolating the intact ER is extremely difficult because of dynamic
transitions in ER composition and its complex association with other organelles and cytosolic
components in live cells; these factors can result in substantial loss of transiently ER-associated
proteins and contamination with other organelles.7
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Chemoproteomic approaches that do not require any subcellular fractionation are
potentially a valuable alternative for circumventing these problems.9–12 Indeed, proximity
biotinylation with ascorbate peroxidase (APEX), a method recently developed by Ting’s group, has
proven to be a powerful tool for organelle proteomics.13–16 In this method, APEX is genetically
localized to a specific organelle, such as the mitochondria or ER. Therefore, the enzymatic oxidation
of the APEX substrate, biotin-phenol, only occurs in a limited space surrounding APEX proteins
upon addition of hydrogen peroxide (H2O2), such that the targeted organelle’s proteins are selectively
modified with a biotin affinity tag. The biotinylated proteins can subsequently be enriched with avidin
beads and identified by liquid chromatography tandem-MS (LC-MS/MS) analysis. Despite the
usefulness and robustness of this technique, APEX technology necessitates expression of an
exogenous engineered peroxidase and the addition of cytotoxic H2O2 to initiate the labeling reaction,
which may potentially cause artificial perturbation of natural biological contexts.17 In contrast, we
recently reported mitochondria- and nucleus-focused proteomics using newly developed
organelle-localizable reactive molecules (ORMs), which need neither genetic manipulation nor
cytotoxic reagents.18,19 ORMs, composed of an organelle-localizing motif and a reactive group,
spontaneously accumulate within the targeted organelle and the resultant high concentration of ORMs
facilitates spatially limited modification of proteins with a detection/purification tag via simple
intermolecular reactions; no specific enzyme activity or protein affinity is required. ORM labeling
using nuclear- or mitochondria-localizable reactive molecules (NRMs and MRMs) combined with
antibody-based enrichment and MS-fingerprinting techniques allows for selective identification of
nuclear and mitochondrial proteomes, respectively.
Here, we sought to challenge the current knowledge of ER-focused proteomics, using an ORM
strategy that exploited a set of ER-localizable reactive molecules (ERMs) exhibiting a variety of
chemical reactivities and amino acid selectivities (Figure 1a). These designer ERMs were observed
to selectively localize within the ER of live cells, which allowed spatially restricted chemical
modification and identification of ER-associated proteins without biochemical fractionation or
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genetic manipulation. LC-MS/MS analysis of the labeled proteins validated the ER selectivity of this
method. Notably, three proteins were newly assigned as ER-related proteins. We also demonstrated
simultaneous usage of three probes, an NRM, MRM, and ERM, in the same cell culture, which
highlights the feasibility of applying our chemical fractionation approach to multiple subcellular
organelles within a single biological sample. Finally, quantitative MS analysis coupled with one of
the ERMs was conducted to analyze the dynamic changes in the ER proteome induced by ER stress.
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Results and discussion
a
Spontaneous
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ER-selective protein labeling ꢀ
Protein IDꢀ
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(2) Digestion
(3) LC-MSMSꢀ
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Figure 1. ER-focused chemical proteomics with a set of ER-localizable reactive molecules (ERMs).
(a) Schematic of ERM-mediated selective labeling and profiling of ER-associated proteins. (b)
Molecular design of ERM 1–6 with the different reactive groups. The calculated logP (clogP) value
for each of these compounds is shown in brackets.
Molecular design of ER-localizable reactive molecules
According to the design principle used in our previous studies,18,19 we prepared a set of
ERMs consisting of an ER-localizable motif and a reactive group (Figure 1b). Fluorinated
hydrophobic rhodol was chosen as a promising ER localizer.20 This fluorescent dye is reported to
specifically accumulate in the ER, presumably because its hydrophobic and amphipathic properties
confer a preference for the environment of the cholesterol-poor ER membranes.21 Furthermore, rhodol
derivatives exhibit excellent photophysical properties that would prove useful for evaluating the
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subcellular localization and concentrations of ERMs by confocal laser scanning microscopy (CLSM)
imaging and spectroscopic in-gel analysis. For the reactive groups, we employed three different
electrophiles, chloroalkane (CA; 1, 2, and 5), sulfonyl fluoride (SF; 3), and thiophenyl ester (TE; 4
and 6). We and other groups previously demonstrated that CA and TE preferentially react with the
thiol group of cysteines and the amino group of lysines, respectively, while SF shows good reactivity
towards both lysines and tyrosines.18, 22–25 Therefore, ERMs equipped with these electrophiles should
exhibit distinct amino acid preference, which may result in different protein selectivity and enhanced
the coverage of proteins in the ER proteome. Given the plausible mechanism of ER-localization with
rhodol, the hydrophobicity of the ERMs was deemed likely to be critical for spontaneous
accumulation in the ER. Thus, ERM derivatives bearing a benzene-type spacer between the rhodol
and the reactive group were also prepared to modulate their hydrophobicity. Figure 1b summarizes
the set of ERMs used together with their calculated logP values (clogP) as an indicator of their
hydrophobicity. Synthesis of the ERMs was carried out as shown in Figure 2 and in the Supporting
Information (SI). ERM 1, for instance, was synthesized as follows: the core compound 16 was
conjugated with a Boc-protected form of the diamine linker through Buchwald–Hartwig
cross-coupling. After deprotection of the Boc group, N-(chloroacetoxy)succinimide was condensed
to yield ERM 1. All the final compounds were characterized by NMR and high-resolution MS
spectroscopy. UV-Vis and fluorescence spectra of the representative ERM 6 are shown in
Supplementary Figure S1.
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Figure 2. Synthetic schemes for ERM 1–6.
Cell permeability and localization of the ERMs
We first evaluated the cell permeability and ER localizability of the ERMs (1–6). HeLa
cells were incubated in culture medium containing each of the ERMs (100 nM) and ER-Tracker Red,
a conventional ER staining dye, at 37 °C for 15 min, followed by CLSM analysis. It was found that
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all ERMs except 2 rapidly passed through the cell membrane (< 15 min) and spontaneously localized
to the ER (Figure 3a and Supplementary Figure S2). The relationship between the ER-selective
localization and the hydrophobicity of the ERMs was examined by plotting the Pearson’s correlation
coefficients (PCC) of the ERM/ER-Tracker Red signal overlap against the clogP values of the
cell-permeable ERMs (Figure 3b). To our surprise, it was clear that (i) the clogP values were
strongly correlated with the ER localization of the ERMs, and (ii) the appropriate lipophilicity (clogP
> 3.4) was required for ER-specific targeting. Indeed, we noticed that the less hydrophobic ERM 1
(clogP = 1.7) was distributed throughout non-ER regions, such as the cytosol and nucleus. We also
found that similar non-specific distribution occurred in cases where high concentrations of ERM 3–6
were used (> 1 µM) (Supplementary Figure S3). Thus, 100 nM of ERM (the apparent concentration
in the medium) was defined as the optimal concentration for ER proteome-specific labeling.
The ER selective localization of ERMs was further evaluated by imaging the colocalization of
ERM 6 with other organelle markers, including Mito-tracker, Lyso-tracker, and CellLight Golgi-RFP.
As shown in Supplementary Figure S4, the fluorescent signal of 6 did not merge with those of
Mito-tracker and Lyso-tracker, clearly indicating the ERM does not localize in mitochondria and
lysosomes. On the other hand, the image of 6 was partially overlapped with that of Golgi apparatus. It
was considered that this is attributed to the vesicle transport of ERM from ER to Golgi apparatus
during this experiment. Indeed, we confirmed that the colocalization of the ERM in Golgi apparatus
was suppressed by addition of H89, a protein kinase inhibitor H89 which can block ER-to-Golgi
trafficking. These indicated that ERMs are precisely localized in the ER at first, and its small portion
gradually translocates to Golgi via ER-to-Golgi transport system during incubation.
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We next quantitatively determined the intracellular concentrations of ERMs by
spectroscopic analysis of the lysates prepared from cells treated with 100 nM of each ERM. The
concentrations of 1–6 per single cell were estimated as shown in Figure 3c. Because the ER
comprises about 3–15% of the cellular volume,26,27 it is deduced that the condensation effect in the
ER further raises intra-organellar concentrations by 6.6~33-fold, which would be reasonably expected
to facilitate the intermolecular ERM-labeling reactions with proteins in the ER (Supplementary
Table S1). Compared with the thioester-type ERM 4 (4.3 µM per cell), the intracellular concentration
of the more hydrophobic thioester-type ERM 6 was 3.5-fold higher (14.9 µM), which suggests that
probe hydrophobicity is one of the key factors both for localization and condensation of ERM within
the ER. Given their excellent ER localizability and reaction diversity, ERMs 3, 5, and 6 were selected
for subsequent protein labeling experiments.
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Figure 3. Cell permeability and spontaneous ER localizability of the ERMs 1–6. (a) Representative
CLSM merged images of HeLa cells treated with 100 nM of each ERM (green) and ER-Tracker (red)
for 15 min. Colocalization areas are in yellow. Line-plot graphs indicate the fluorescence intensity
profiles of the ERM (green) and ER-Tracker (red) along the white dotted lines indicated in the CLSM
images. Scale bar: 20 µm. (b) Correlation of the Pearson’s correlation coefficients of the
ERM/ER-Tracker signal overlap observed in the CLSM images against the clogP values of the
cell-permeable ERMs (1, 3–6). (c) Intracellular concentration of each ERM in HeLa cells determined
by spectroscopic measurement of rhodol fluorescence.
Protein labeling with ERMs
Once the three optimal ERMs (3, 5, and 6) for selective ER localization had been
determined, we next evaluated the reactivity of these ERMs with endogenous ER proteins in HeLa
cells using CLSM imaging and gel electrophoresis. After incubation of the cells with each ERM for 1
h, the cells were fixed with cold methanol to remove unreacted ERMs, followed by CLSM analysis.
As shown in Figure 4a, strong fluorescent signals from rhodol remained for all three ERMs even
after methanol fixation, and more importantly, this fluorescence merged well with the
ER-Tracker-based signal.28 In contrast, no fluorescence was detected for control compound 7, which
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lacked a reactive group.29 This ER-localized fluorescence clearly indicated that the ERMs selectively
accumulated within the ER and labeled the ER-localized proteins with the rhodol dye, providing
stable fluorescence signal in the ER even after cell fixation. The covalent attachment of rhodol to the
ER proteins was additionally confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
followed by in-gel fluorescence imaging (Figure 4b and Supplementary Figure S6). On these gels,
many fluorescent bands were detected in all three lanes where each of the ERMs were separately
applied to the HeLa cells. Notably, the band patterns for the three ERMs were distinct. These results
confirmed covalent bond formation between the ER proteins and the rhodol dye, as well as the
different protein selectivity of each ERM.
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PCC = 0.837ꢀ PCC = 0.884ꢀ PCC = 0.945ꢀ
3ꢀ 5ꢀ 6ꢀ
Reagentꢀ
Figure 4. Covalent protein modification in the ER with ERMs. (a) CLSM images of HeLa cells
treated with ERMs 3, 5, 6, and 7 (green) and ER-Tracker (red), followed by methanol fixation.
Colocalization was quantified using Pearson’s correlation coefficient (PCC). Scale bar: 5 µm. (b)
SDS-PAGE analysis with in-gel fluorescence imaging. Reaction conditions: HeLa cells were
incubated with each ERM (100 nM) in DMEM at 37 °C for 1 h. After cell lysis, the labeled proteins
were enriched by immunoprecipitation with anti-rhodol antibody to enhance the fluorescent signal,
followed by SDS-PAGE (a 4-12% gradient gel). Optimization of the reaction time with each ERM in
living cells is shown in Supplementary Figure S6.
ER protein identification and assignment of new ER proteins
Identification of proteins labeled with each ERM was performed with LC-MS/MS analysis
after antibody-based enrichment (Figure 5a). We prepared anti-rhodol antibody in-house and
confirmed that the antibody was applicable for the enrichment experiments, using isolation of
rhodol-modified proteins from cell lysates by immunoprecipitation (IP) (Supplementary Figures S7
and S8). The enriched proteome was then subjected to SDS-PAGE followed by in-gel tryptic
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digestion, and the resultant peptide fragments were analyzed by nano-LC-MS/MS. The annotation
analysis of the cellular compartments occupied by the identified proteins was carried out using the
latest UniProtKB, the Gene Ontology Cell Component database and ProteinCenter web-based
software (Thermo Scientific).30,31 Figure 5b and Supplementary Data S1 summarize the identified
proteins and their localizations. Using ERM 3 as the labeling reagent, 51 proteins were identified,
among which seven proteins were assigned as ER proteins (ER ratio: 13.7%) based on the database
analysis. Similarly, 19 (among 68), and 28 (among 146) ER proteins were identified by 5 and 6,
respectively. In total, 195 proteins were identified using the three ERMs, which included at least 41
ER proteins (ER ratio: 21%).32 The relatively low ER ratio observed might have been due to the
incomplete database set of ER-associated proteins. Thus, we decided to manually verify the cellular
localization of the identified proteins through a literature survey (see Supplementary Data S1).
Using this approach, another 43 proteins (22.1%) were reassigned as ER-associated proteins.
Consequently, the total ER ratio was improved to 43.1%.33,34 It should be noted that among the
remaining 56.9% not assigned as ER proteins, 46.6% were proteins with cellular component
annotations derived from the ER, that is, ‘membrane’, ‘Golgi’, and ‘extracellular space’ (Figure
5b).35 These data suggest that the latter set of proteins were labeled by the ERMs before being
recruited to their final destinations. We further noticed that a considerable number of secretory
proteins were included in our dataset. This is very reasonable, given that the ER functions as a central
hub for vesicle transport and the secretory pathways of proteins in live cells.2 A similar trend was
discussed in a recent study by Ting’s group.15 We therefore next interrogated the obtained proteome
for proteins with secretory pathway annotation, which indicated that 92% of the total number of
proteins identified by the ERMs were associated with secretory pathways (that is, 180 out of 195
proteins identified in total could reasonably be assigned as ER-related proteins) (Figure 5c). This
value is considerably high compared with the known proportion of these proteins in the entire human
proteome (51%; 9,731 out of 18,962 proteins)15, which clearly reveals that ER-related secretory
proteins were efficiently enriched by our ERM-based method.
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We further sought to validate the localization of several of the proteins that did not previously
have ER annotation but were identified by ERMs as ER-associated proteins (that is, proteins
identified in this study and annotated their localization as Membrane/Golgi/Extracellular in Figure
5b). Four such proteins were identified: multifunctional protein ADE2 (PAICS), thioredoxin-like
protein 1 (TXNL1), peptidyl-prolyl cis-trans isomerase A (PPIA), and D-3-phosphoglycerate
dehydrogenase (PHGDH). We performed co-immunostaining of these five proteins with the
respective antibody and the ER marker protein, calnexin (CNX). As shown in Figure 5d,
immunofluorescence signals from PAICS, TXNL1, and PPIA were detected in the ER, suggesting
that these proteins are potentially involved in ER function. PAICS is a bifunctional enzyme
containing phosphoribosylaminoimidazole carboxylase activity in its N-terminal region and
phosphoribosylaminoimidazole-succinocarboxamide synthase activity in its C-terminal region.36
This enzyme is also a member of the purine biosynthesis pathway. Previous studies indicated that
PAICS is associated with extracellular vesicles and the cell membrane, features consistent with an
ER-associated protein.37,38 TXNL1 shows protein-disulfide reductase activity and may be involved in
protein folding in the ER.39,40 PPIA is a chaperone protein that catalyzes cis-trans isomerization of
proline imide peptide bonds and is known to be secreted in response to oxidative stress.41,42 It is
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therefore reasonable for this protein to pass through the ER. PHGDH did not exhibit good
immunofluorescence overlap, presumably because it represents false positive or it is present in a low
amount in the ER (Supplementary Figure S12). Overall, although further investigation regarding the
localization of these proteins is required, the results obtained here highlight the usefulness of
ERM-based chemical proteomics for identifying new ER-related proteins.
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Enrichment of
rhodol-labeled proteins
Labeled proteome
Protein IDꢀ
IP with
anti-rhodol antibodyꢀ
Digestionꢀ
LC-MSMSꢀ
m/zꢀ
m/zꢀ
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(8%) ꢀ
c
Unassignedꢀ
Other organelles/cytosolꢀ
Membrane/Golgi/Extracellularꢀ
ER (manually assigned)ꢀ
ER (database)ꢀ
1.5%ꢀ
8.7%ꢀ
180
(92%) ꢀ
1.4%ꢀ
5.5%ꢀ
Secretory
ERM proteomeꢀ
46.7%ꢀ
50%ꢀ
Non-secretory
1.5%ꢀ
7.3%ꢀ
41.1%ꢀ
22.1%ꢀ
21%ꢀ
9731
(51%) ꢀ
9231
(49%) ꢀ
13.7%ꢀ
23.9%ꢀ
19.2%ꢀ
52.9%ꢀ
22.1%ꢀ
28%ꢀ
19.6%ꢀ
13.7%ꢀ
Entire human proteomeꢀ
Enlargedꢀ
d
PAICSꢀ
TXNL1ꢀ
PPIAꢀ
ER (CNX)ꢀ
ER (CNX)ꢀ
ER (CNX)ꢀ
Mergeꢀ
Mergeꢀ
Enlargedꢀ
Mergeꢀ
Enlargedꢀ
Figure 5. ERM-based identification and profiling of rhodol-labeled proteins in the ER. (a) Schematic
of the approach used in this study for protein identification. The rhodol-labeled proteins were
enriched by IP with anti-rhodol antibody and then identified by LC-MS/MS analysis. (b) The number
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of proteins identified in HeLa cells using the ERM-based approach, and the ratio of proteins with ER
annotations to proteins with other annotations. The identified proteins were classified as ‘ER’,
‘membrane/Golgi/extracellular’, ‘other organelles/cytosol’ or ‘unassigned’ according to the
UniProtKB database, the Gene Ontology (GO) cell component annotation terms and manual literature
surveys. Detailed information on the identified proteins is presented in Supplementary Data S1. (c)
Pie charts of the percentage of identified proteins with secretory pathway annotation according to the
GO database or the Phobius predictive tool (Ref.43) among proteins identified with our method (top)
and reported for the entire human proteome (bottom). (d) Subcellular localization of the three proteins,
PAICS, TXNL1 and PPIA, newly assigned to the ER in this study. HeLa cells were fixed and stained
with anti-PAICS, anti-TXNL1 and anti-PPIA (green), and anti-calnexin (CNX) (red) to visualize the
ER. Colocalization regions are shown in yellow in the merge images. Scale bar: 5 µm.
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Simultaneous use of nuclear-, mitochondria-, and ER-localizable reactive molecules
We next sought to multiplex the ERMs together with other ORMs such as the
Hoechst-based
NRMs
(nucleus-targeted)
and
the
tetraethylrhodamine-based
MRMs
(mitochondria-targeted) (Figure 6a).18,19 These probes were expected to orthogonally localize to their
respective target subcellular compartments in the same biological sample and to modify their target
organellar proteins with a distinct dye. After cell lysis, the labeled proteins could be enriched by IP
using specific antibodies against Hoechst, tetraethylrhodamine, and rhodol, and sorted into nuclear,
mitochondrial, and ER-associated proteins, respectively. Thus, the chemical labeling-mediated
isolation of proteins from each of these three organelles would be achieved without subcellular
fractionation.
We first tested the orthogonal localization of NRM, MRM, and ERM in live cells. HeLa
cells were simultaneously treated with ERM 6 (0.1 µM), NRM 8 (5 µM), and MRM 9 (0.5 µM) and
subsequently analyzed with Zeiss Airyscan super-resolution microscopy. Thanks to the distinct
fluorescence wavelength of each of these localization moieties, the nucleus, mitochondria, and ER
were clearly and specifically stained by NRM 8, MRM 9, and ERM 6, respectively, demonstrating
that these three probes exclusively localize and concentrate within their target organelle even when
applied together (Figure 6b and Supplementary Figure S13). We further confirmed that both NRM
8 and MRM 9 did not label proteins in the ER (Supplementary Figure S14).
Encouraged by the imaging data, we proceeded to characterize the labeled proteins by
protein enrichment combined with LC-MS/MS analysis. We employed a sequential IP protocol for
the selective isolation of tagged proteins (Figure 6c). In the process of optimization for the iterative
IP, it was found that anti-tetraethylrhodamin and anti-rhodol antibodies crossreact with Hoechst and
tetraethylrhodamine molecules, respectively (Supplementary Figure S15). Thus, to sidestep the
crossreaction, the lysates of labeled cells were successively subjected to IP with anti-Hoechst,
anti-tetraethylrhodamine, and anti-rhodol in that order, followed by MS fingerprinting analysis. We
identified 56 and 282 proteins within the NRM and MRM fractions, and 44 (79%) and 223 (79%) of
these proteins were assigned as nuclear and mitochondrial proteins, respectively, which was
consistent with results from our previous study on the separate use of this NRM and MRM (Figure
6d and Supplementary Data S2).18,19 While the number of identified proteins in the ERM fraction
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was less than when it was separately used, mainly because of protein loss during the iterative IP steps,
the ER ratio remained high (42%) (89% had secretory pathway annotation). These results show the
potential of this approach for orthogonally tagging and identifying organellar proteins by using
multiple organelle-localizable reactive molecules in a single sample.
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N
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H
N
ERM 6
(ER)ꢀ
O
O
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H
Cl
3
O
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N
O
N+
NH
N
O
NRM 8
(Nucleus)ꢀ
N
NH
N
N
N
Cl
O
MRM 9
(Mitochondria)ꢀ
b
NRM 8ꢀ
MRM 9ꢀ
Merge
ERM 6ꢀ
c
d
2nd IP with
3rd IP with
anti-Rhodamineꢀ anti-Rhodolꢀ
Simultaneously
1st IP with
add 6, 8, 9
anti-Hoechstꢀ
NRMꢀ MRMꢀ
ERM
HeLa cellsꢀ
Target
ER: 15
ꢁ4ꢀ
56ꢀ
79ꢀ
223ꢀ
282ꢀ
79ꢀ
organelleꢀ
Secretory: 32ꢀ
Wholeꢀ
%ꢀ
36ꢀ
ER: 42
1. in-gel digestion
2. LC-MSMSꢀ
Secretory: 89ꢀ
ER
Nuclear
protein ID
Mitochondria
protein ID
protein ID
Figure 6. Simultaneous use of NRM, MRM, and ERM for targeted protein enrichment in HeLa cells.
(a) Molecular structures of ERM 6, NRM 8, and MRM 9. The reaction properties of 8 and 9 are
reported in references 19 and 18, respectively. (b) CLSM images of a live HeLa cell concurrently
treated with ERM 6 (100 nM, green), NRM 8 (5 µM, blue), and MRM 9 (0.5 µM, red). (c) Workflow
of the sequential IP protocol for the enrichment of proteins labeled with each reagent. (d) The cellular
locations of the identified proteins were analyzed according to the protein ontology information
provided in the UniProt database and from manual literature surveys.
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Quantitative profiling of ER-proteins during tunicamycin-induced unfolded protein response
ER stress, which is characterized by an increase in unfolded proteins in the ER, results in
the activation of specific cellular responses such as the unfolded protein response (UPR) and
ER-associated protein degradation (ERAD).44,45 We applied our ERM-based proteomics approach to
examine the dynamic changes in the levels of different ER-associated proteins in response to ER
stress. In this experiment, the ERM-based protein tagging strategy was combined with stable isotope
labeling by amino acids in cell culture (SILAC) technology for quantitative analysis of the ER
proteins during tunicamycin (Tm)-induced UPR (Figure 7a).46–48 HeLa cells grown in heavy SILAC
media were treated with Tm (2.5 µg/mL) for 4 h, followed by treatment with ERM 6 (100 nM, 1 h),
whereas cells grown in light SILAC media were treated with ERM 6 only. The presence of
Tm-induced UPR was verified based on the expression of CHOP, a representative UPR marker
protein (Supplementary Figure S16).46,47,49 We also confirmed that the ERM localized in the ER of
the Tm-treated cells as well as the normal cells and that the intracellular concentration of the ERM
did not substantially change following Tm treatment (Supplementary Figure S17). As shown in
Supplementary Figure S18, the fluorescent band patterns on the SDS-PAGE gels were almost
identical between the Tm-treated and nontreated cells, indicating that Tm-induced UPR did not
largely alter the expression of most ER proteins in HeLa cells in our protocol. The lysates of light-
and heavy-labeled cells were mixed in a 1:1 ratio and the rhodol-tagged proteins were enriched as
described above, followed by LC-MS/MS analysis. ERM 6 identified and quantified 87 proteins
across three independent biological replicates (Figure 7b and Supplementary Data S3). Among
these 87 proteins, 39 proteins (45%) were classified as ER proteins by our annotation analysis, while
84 proteins (97%) were assigned with secretory pathway annotation. Of the identified proteins, six
ER proteins were upregulated by more than two-fold in Tm-treated cells, while only one
ER-associated protein (HSP90AB) displayed significantly decrease in the log2 value (Figure 7b).
Notably, three well-known UPR-related proteins, GRP78, HSP60, and HO2, were included in the six
up-regulated proteins. GRP78 is one of the major UPR marker proteins and is reported to be
upregulated under UPR conditions.50 HSP60 is classically defined as an intra-mitochondrial
chaperone protein, but it was recently revealed that this protein can also be released via the ER/Golgi
protein secretory pathway.51 Indeed, we confirmed that HSP60 surely exists in the ER of HeLa cells
and is included in the proteins pull-downed by IP using anti-rhodol antibody (Supplementary Figure
S19). According to previous reports, the expression level of HSP60 slightly increases in response to
UPRs.52,53 We verified the increased expression levels of GRP78 and HSP60 by western blotting
analysis (Figures 7c and 7d). Given the recent study suggesting the linkage between HO2-mediated
reactive oxygen species (ROS) suppression and ER stress, up-regulation of HO2 may suppress
UPR-induced apoptosis.54 These results clearly demonstrated that the present ERM-based method can
be successfully applied to the sensitive analysis of dynamic ER proteome changes.
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ERM 6ꢀ
a
b
Tmꢀ
4 hꢀ
Quantitative
MS analysisꢀ
IP
with
anti-rhodol
Mix
lysate
(1 : 1)ꢀ
Tm-
treatedꢀ
in-gel
digestionꢀ
SILAC
“Heavy”ꢀ
Heavyꢀ
labelingꢀ
Lightꢀ
vehicleꢀ
Controlꢀ
SILAC
“Light”ꢀ
9
m/zꢀ
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Tm
–
+ꢀ
c
d
GRP78ꢀ
Txn ꢀ
HSP60ꢀ
HSP60ꢀ
RPL11 ꢀ
VDAC3ꢀ
5ꢀ
4ꢀ
3ꢀ
2ꢀ
1ꢀ
0ꢀ
RPL28 ꢀ
HO2ꢀ
GRP78ꢀ
HIST1H3A
HSP90AB
Proteinꢀ
Figure 7. SILAC-based quantitative MS analysis of ERM proteome change induced by ER stress. (a)
Design of SILAC experiments coupled with ERM-mediated protein labeling to quantify ER proteome
changes in tunicamycin (Tm)-treated HeLa cells. Tm-treated cells were grown in heavy SILAC
medium and nontreated cells were grown in light SILAC medium (control), followed by labeling with
ERM 6. After cell lysis, the lysates from the light and heavy cells were mixed in a 1:1 ratio and the
rhodol-labeled proteins were enriched by IP using an anti-rhodol antibody. The enriched proteins
were subjected to in-gel tryptic digestion and quantitative LC−MS/MS analysis. (b) Log2 ratio plot
(Tm-treated cells (heavy)/control cells (light)) of the identified proteins. Proteins more than 2-fold
upregulated and the others are shown in red and black in the plot, respectively. Gene names written in
green or black letters are proteins assigned as ER-associated proteins (assigned by database and
literatures) or non-ER proteins, respectively. (c) Western blotting analysis of Tm-induced changes in
the expression levels of GRP78 and HSP60. (d) Relative intensities of the western blotting analysis
shown in (c). Data represent the mean ± SD, n = 3.
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Conclusions
1
We developed a set of ERMs that spontaneously accumulate in the ER of live cells and selectively
label ER-localized proteins with an enhanced concentration effect. ERM-based protein tagging allows
the selective enrichment and quantitative profiling of ER-associated proteins without any
time-consuming and technically difficult ER fractionation steps. This method needs neither genetic
manipulation nor harsh labeling conditions. More importantly, the present study demonstrated that
ERMs have the potential to discover unknown ER-related proteins. However, given that ER
proteomes of 1,190 and 1,888 proteins are registered in the UniProt and Gene Ontology databases,
respectively, the present ERM-based method still only covers a small fraction of ER proteins. As
discussed previously,18 this limited coverage, in other word, false negatives may be attributed to (i)
biased sub-ER distribution of the rhodol-based ERMs (in the ER-membrane), and/or (ii) failure to
label proteins that do not have suitable reactive nucleophilic amino acids on the protein surface.
These limitations could be overcome by varying the localization moiety to target different areas
within the ER and by employing a variety of reactive groups exhibiting distinct amino acid
selectivities. In this regard, the linear relationship we observed between the clogP values of the
probes and their ER localization may assist the future rational design of a variety of ER-localizable
reagents. We also demonstrated the feasibility of the simultaneous use of three organelle-localizable
reactive molecules, which should prove a powerful tool for monitoring proteome changes across
multiple organelles in the same cell culture. We believe that the extension of our chemical strategy to
other subcellular organelles, such as the lysosome, Golgi apparatus, and peroxisome, will facilitate
further elucidation of the complicated organellar protein networks in living cells.
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Supplementary information
Supplementary figures and tables, experimental procedures, synthesis and characterization of
compounds. Supplementary Data S1–S3. Raw data for all LC-MSMS analysis.
Author information
Corresponding Author
*E-mail: ihamachi@sbchem.kyoto-u.ac.jp.
ORCID
Tomonori Tamura: 0000-0003-1648-9296
Itaru Hamachi: 0000-0002-3327-3916
Acknowledgements
We thank Karin Nishimura (Kyoto University) for experimental support of MS measurements, and
Prof. Fumiyoshi Ishidate and Prof. Takahiro Fujiwara (Center for Meso-Bio Single-Molecule Imaging,
Kyoto University) for experimental support of super-resolution microscopy. We also thank Natasha
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Beeton-Kempen, Ph.D., from Edanz Group (www.edanzediting.com/ac) for editing a draft of this
manuscript. This work was supported by Grant-in-Aid for Young Scientists (B) (15K17884) and the
Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology
(CREST) to I.H. This work was also supported by a Grant-in-Aid for Scientific Research
on Innovative Areas“Chemistry for Multimolecular Crowding Biosystems”(JSPS KAKENHI Grant
No. 17H06348).
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Competing financial interests
The authors declare no competing interests.
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that the content of the ER proteins decreased to only 17 or 11%, respectively (Supplementary
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3
O
O
H
O
O
I
H2N
Boc
O
F
O
N
Boc
4
N
Cl
N
H
N
H
O
5
6
7
8
9
O
F
F
F
Pd(OAc)2, Xantphos, Cs2CO3
Toluene
TFA
CH2Cl2
Et3N
dry DMF
1
17
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O
O
O
H
N
O
O
O
HO
H
N
H
H
N
Cl
N
O
N
Boc
F
F
Boc
O
EDCI-HCl, HOBt-H2O
DIEA
TFA
TFA
Et3N
17
2
dry DMF
CH2Cl2
CH2Cl2
7
dry DMF
O
F
S
O
Cl
O
TFA
Et3N
7
3
dry DMF
CH2Cl2
H
N
O
F
O
COOMe
COOH
Thiophenol
H2N
F
Pd(OAc)2, Xantphos, Cs2CO3
Toluene
HATU, DIEA
dry DMF
16
4
18
O
Cl
Cl
TFA
DIEA
7
5
dry THF
CH2Cl2
H2N
H
N
H
N
O
O
F
O
O
OEt
Thiophenol
O
F
OEt
NaOH
MeOH
HATU, DIEA
HATU, DIEA
dry DMF
6
18
dry DMF
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In-gel
fluorescenceꢀ
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Journal of the American Chemical Society
a
b
(kDa)ꢀ
250 -ꢀ
3ꢀ
5ꢀ
6ꢀ
7 (control)ꢀ
1
2
3
4
100 -ꢀ
75 -ꢀ
5
6
ERꢀ
ERꢀ
ERꢀ
ERꢀ
50 -ꢀ
37 -ꢀ
7
8
9
10
11
12
13
14
15
16
17
25 -ꢀ
15 -ꢀ
Mergeꢀ
Mergeꢀ
Mergeꢀ
Mergeꢀ
ACS Paragon Plus Environment
PCC = 0.837ꢀ PCC = 0.884ꢀ PCC = 0.945ꢀ
3ꢀ 5ꢀ 6ꢀ
Reagentꢀ

Journal of the American Chemical Society
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H
N
H
N
a
O
O
O
O
F
F
S
O
1
2
3
4
5
H
N
ERM 6
(ER)ꢀ
O
O
N
H
Cl
3
O
N
N
O
N+
NH
6
7
8
9
N
O
NRM 8
(Nucleus)ꢀ
N
NH
N
N
N
Cl
O
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11
12
13
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18
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20
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24
25
MRM 9
(Mitochondria)ꢀ
b
NRM 8ꢀ
MRM 9ꢀ
Merge
ERM 6ꢀ
c
d
26
2nd IP with
3rd IP with
Simultaneously
1st IP with
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anti-Rhodamineꢀ anti-Rhodolꢀ
add 6, 8, 9
anti-Hoechstꢀ
NRMꢀ MRMꢀ
ERM
HeLa cellsꢀ
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Target
organelleꢀ
ER: 15
Secretory: 32ꢀ
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34
35
36
37
38
39
40
ꢁ4ꢀ
56ꢀ
79ꢀ
223ꢀ
282ꢀ
79ꢀ
Wholeꢀ
%ꢀ
36ꢀ
ER: 42
Secretory: 89ꢀ
1. in-gel digestion
2. LC-MSMSꢀ
ACS Paragon Plus Environment
ER
protein ID
Nuclear
protein ID
Mitochondria
protein ID