Cell type-selective imaging and profiling of newly synthesized proteomes by using puromycin analogues

Article abstract of DOI:10.1039/c7cc04536k

We have developed a versatile antibody-assisted strategy for the imaging and profiling of newly synthesized proteomes in a cell-specific manner. This strategy remained highly selective even in heterogeneous co-cultured cells, thus enabling labeling and enrichment of nascent proteomes from targeted cells without the need for physical separation.

Full text of DOI:10.1039/c7cc04536k

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DOI: 10.1039/C7CC04536K
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Cell Type-Selective Imaging and Profiling of Newly Synthesized
Proteomes by Using Puromycin Analogues
Shubo Du,a Danyang Wang,a Jun-Seok Lee,b Bo Peng,a Jingyan Gec* and Shao Q. Yaoa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We have developed a versatile antibody-assisted strategy for genetic modifications, however, renders it to have limited
imaging and profiling of newly synthesized proteomes in a cell- applications in tissues or whole organisms in their native state.
specific manner. This strategy remained highly selective even in The second strategy involves the selective activation of a caged
heterogeneous co-cultured cells, thus enabling labeling and protein tag (e.g. puromycin) by removing the blocking group
enrichment of nascent proteomes from targeted cells without the through a cell-specific enzyme.7,11 Puromycin (PO) is an
need for physical separation.
aminonucleoside antibiotic which could be used as a tag for
synthesis of nascent polypeptides.12 Recently, multiple PO
analogues were developed to achieve multiplexed imaging and
profiling of protein synthesis in live cells and neurons.13 Since
PO can function as a protein tag at a much lower concentration
than noncanonical amino acids,7,12,14 low-abundance enzymes
might be employed as deblocking triggers. Moreover, PO
provides a better temporal resolution than that of noncanonical
amino acids.12,15 In order to achieve cell-selective incorporation
of PO into nascent polypeptides, a chemical genetic method
was developed that involves an engineered enzyme to activate
a clickable PO analogue.7 However the deblocking enzyme was
not endogenous, and the strategy was restricted to one specific
enzyme. It is often the case that decaging triggers and blocking
groups must be optimized for each new target cell population
in order to maximize the PO release. Given the caveats of each
of these methods, there remains a critical need to develop a
more versatile and robust PO-based strategy to label newly
synthesized proteomes from specific cells at a specific time.
Inspired by the success of antibody-drug conjugates (ADC),
we turned to antibodies as potential decaging triggers in our
cell-selective proteome labelling strategy (Figure 1).16 In order
to achieve the release of PO needed to tag nascent
polypeptides, we took advantage of the inverse-electron-
demand Diels–Alder (inv-DA) reaction, a “click-to-release”
approach that affords instantaneous payload release upon
conjugation between trans-cyclooctene (TCO) and tetrazine
Global proteome analyses have provided important insights
into diverse biological processes. Metabolic labelling of proteins
with unnatural amino acids permits the analysis of the
spatiotemporal regulation of protein synthesis.1 However,
metabolic incorporation strategies are generally nonspecific
with respect to cell identity, rendering them of limited use in
heterogeneous cellular systems.2-5 Differentiating the various
proteomes of individual cell subpopulations from co-cultures is
highly important in cell-cell communications, e.g. host-
pathogen interaction and intercellular signal transduction.2,6
Therefore it is highly desirable that cell-selective methods are
developed to resolve proteome changes with sufficient
spatiotemporal resolutions. However, the study of cell-specific
translational regulation in vivo is challenging, mainly due to the
difficulty to ascertain the origin of the identified proteins in
multi-cell cultures, especially for low-abundance cell
populations.7 Two major strategies have been exploited to
address this issue. The first is the use of engineered aminoacyl-
tRNA synthetases that specifically recognize and incorporate
noncanonical amino acids into transgenic cells.8-10 The need for
a.S. Du, D. Wang, B. Peng, Prof. Dr. S. Q. Yao
Department of Chemistry, National University of Singapore.
3 Science Drive 3, Singapore 117543 (Singapore)
E-mail: chmyaosq@nus.edu.sg
b.Prof. Dr. J.-S. Lee
(
Tz).17-19 Instead of directly linking PO to the antibody, we
Molecular Recognition Research Center & Department of Biological Chemistry,
KIST-School Korea Institute of Science & Technology (South Korea)
c. Dr. J. Ge
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of
Biotechnology and Bioengineering, Zhejiang University of Technology (China).
E-mail: gejy@zjut.edu.cn
modified the antibody with Tz to achieve easy antibody
modification, maximize the number of covalent binding sites
and minimize interference to the antigen-antibody interaction.
PO was introduced as TCO-PO in the second step. The TCO
group effectively blocks the free amino group on PO, rendering
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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a)
Journal Name
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DOI: 10.1039/C7CC04536K
TCO-PO ONLY
Cetuximab-Tz + TCO-PO
b)
c)
NIH3T3
CHO-K1
HeLa
+
+
-
MCF-7
A549
+
+
-
A431
+
+
-
TCO-PO
+
+
+
-
-
+
-
+
+
-
-
-
+
+
-
-
-
+
-
-
+
-
+
-
-
-
+
-
-
-
-
+
Cetuximab-Tz -
-
+
-
+
-
-
+
-
-
-
+
+
PO
250
130
100
A431
EGFR(+)
70
55
CHO-K1
EGFR(-)
35
25
Figure 1. a) Antibody-assisted cell type-specific delivery and release of PO with “click-to-release” chemistry in a heterogeneous environment. b) Selective
labelling of nascent polypeptides in EGFR-overexpressing A431 cells against a panel of other cell lines with no/low endogenous EGFR expression. Synthesis of
nascent proteomes was detected by Western blotting (WB) using anti-puromycin antibody. EGFR expression was assessed by WB analysis with anti-EGFR
antibody, and the total proteome was visualized by Ponceau S stain (Figure S4). c) A431 and CHO-K1 cells were sequentially treated with Cetuximab-Tz and
TCO-PO. Labelled proteins were detected by immunofluorescence (IF; green channel) with anti-puromycin. Fluorescence microscopy revealed labelled
proteins in A431 cells, whereas proteins were not labelled in the absence of Cetuximab-Tz or in CHO-K1 cells. (blue): DAPI. Scale bar = 15 μm.
it unable to be incorporated in protein synthesis.
TCO-PO (3 μM) for 24 h resulted in extensive PO labelling across a
As shown in Figure 1a, the Tz-loaded antibody (i.e. spectrum of molecular weights (boxed lane 2 in Figure 1b); in the
Cetuximab-Tz) binds to a tumour cell-specific membrane absence of Cetuximab-Tz, cells were essentially devoid of PO
receptor. Subsequently TCO-PO is introduced. Upon ligation incorporation (lane 1). Importantly, no labelling was observed in
between TCO and tetrazine, the resulting adduct undergoes similarly treated cells known to have no/low EGFR expression (i.e.
conversion into a conjugated pyridazine with the concomitant NIH3T3/CHO-K1/HeLa/MCF-7/A549; Figure 1b and Figure S6). In
elimination of CO2 and release of free PO
S3). Labelled proteins can be distinguished from the rest of the panel of cells directly treated with PO (lanes 3 in Figure 1b). We
protein pool through either immunological staining or further observed concentration-dependent increase in PO
bioorthogonal conjugation that permits facile visualization and incorporation from A431 cells with an increasing amount of TCO-PO
isolation of proteins in a cell specific manner (see Figures 2 ). (Figure S7). Under these conditions, protein labelling was equivalent
(Figure 1a and Figure contrast, robust PO incorporation was observed across the entire
a
&
3
As an example to demonstrate the feasibility of our strategy, to a low dose of PO incorporation previously shown to cause little or
we chose to selectively label the newly synthesized proteomes no disruption to synthesis of newly synthesized proteomes.13,23 XTT
in A431 cancer cells, which are known to endogenously cell viability assay with (Cetuximab-Tz + TCO-PO)-treated A431 cells
overexpress epidermal growth factor receptor (EGFR).20 The showed no obvious difference in cell vitality when compared with
monoclonal antibody Cetuximab, which is used clinically for cells treated with Cetuximab-Tz or TCO-PO individually, indicating
cancer treatment, was used to target EGFR.21 The antibody was that PO treatment under our conditions did not have significant cell
modified with Tz-NHS according to literature with minor toxicity (Figure S8). A431 cells treated with increasing doses of
modifications.22 Tz modifications had little effect on binding Cetuximab-Tz for 30 min showed a significant increase in PO
affinity and did not affect the level of non-specific binding to incorporation, and the labelling can be partially blocked by pre-
control CHO-K1 cells (Figure S2). (E)-cyclooct-2-enol was incubation with unmodified Cetuximab (Figure S9), indicating that
synthesized according to published procedures16 and PO release was dependent on the endogenous EGFR expression
subsequently linked to the α-amino group on PO through level. To further demonstrate the deblocking of TCO-PO occurred
carbamate formation. The reaction between Tz and TCO-PO only in cells expressing a high level of EGFR, we monitored PO
and subsequent release of free PO was confirmed by LC-MS incorporation into nascent polypeptides in single cells by using IF
analysis
(Figure S3). The hydrolytic stabiltiy and cell with anti-puromycin antibody (Figure 1c). In these experiments, the
permeability of TCO-PO was also confirmed (Figure S4 & 5).
cells were similarly incubated with Cetuximab-Tz followed by TCO-
To determine whether Cetuximab-Tz can remove the TCO PO. Strong fluorescence signals were detected in A431 cells, with a
blocking group on TCO-PO in EGFR-overexpressing A431 cells, we uniform distribution in the cytoplasm. These results are similar to
monitored the incorporation of PO into nascent polypeptides by those in cells directly treated with PO (Figure S10). In contrast, no
using Western blotting (WB) with anti-puromycin antibody. fluorescence was observed when Cetuximab-Tz was omitted, or with
Treatment of A431 cells with Cetuximab-Tz (10 μg/mL) followed by CHO-K1 cells. Taken together, our results unequivocally demonstrate
2 | J. Name., 2012, 00, 1-3
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CHO-K1 cells. This resulting co-culture of A431 and CHO-K1 cells were
further seeded in glass-bottom dishes, and cell labelling and IF
View Article Online
DOI: 10.1039/C7CC04536K
a)
imaging experiments were finally carried out (Figure 2b); to our
delight, the antibody binding and PO incorporation were both
restricted to EGFR-expressing A431 cells (arrowed in red), and no
labelling was observed in the co-cultured CHO-K1 cells (arrowed in
blue). The high cell type-specificity could be due to two reasons. The
EGFR-Cetuximab complex was constantly internalized into the cells
(Figure S2c), therefore the decaging process partially occurred
intracellularly. Moreover, even when the decaging occurred on the
cell surface, the decaged PO can be rapidly taken up by cells. Due to
the relatively low expression of cell-surface protein receptors, the
decaged PO was effectively maintained at a low concentration, thus
limiting its diffusion to the medium and neighbouring cells.
IF
EGFR-
mCherry
Overlay
(anti-puro)
1)
2)
3)
EGFR-
expressingCells
-
4)
5)
6)
+
EGFR-free Cells
b)
Having demonstrated the successful labeling of nascent
polypeptides with PO exclusively in EGFR-expressing cells, we next
sought to identify newly synthesized proteomes from target cells by
using our strategy. We first synthesized an alkyne-bearing PO
IF
Mito + Nu
Overlay
(anti-puro)
7)
8)
9)
-
EGFR-
expressing Cells
analogue, TCO-AY (Figures 3a,b
& S12); the release and
10)
11)
12)
incorporation of TCO-AY was confirmed by gel-based profiling and
imaging experiments with Cetuximab-Tz-treated A431 and in
transfected CHO-K1 cells. We next combined this antibody-assisted
proteome labelling strategy with stable-isotope labelling by amino
acids in cell culture (SILAC) and liquid chromatography–tandem mass
spectrometry (LC-MS/MS). As shown in Figure 3c, isotopically ‘‘Light”
cells served as a static control and were treated with TCO-AY only.
Isotopically ‘‘Heavy” cells served as comparison groups and were
treated with both Cetuximab-Tz and TCO-AY. The experiments were
termed as “Forward”. To account for differences in protein
abundance between two isotopically labelled cells, we furthermore
carried out “Reverse” experiments in which the probe treatment was
reversed. The SILAC experiments were performed in both A431 and
CHO-K1 cell lines. After 24-h incubation, CuAAC conjugation with
TMR-biotin-azide was performed in cell lysates, and biotinylated
proteins were enriched using NeutrAvidin beads.24 Eluted proteins
were SDS-PAGE-separated, followed by in-gel digestion and finally
LC-MS/MS. Upon data analysis, proteins with a SILAC Heavy/Light
ratio of ≥ 3 in Forward experiments or ≤ 0.3 in Reverse experiments
were identified as hits. As shown in Figure 3d, we were able to
identify significantly more proteins in (Cetuximab-Tz + TCO-AY)-
treated A431 cells than in similarly treated CHO-K1 cells. Taking into
consideration of the non-specific background labeling and intrinsic
variability of large-scale proteomic profiling experiments, the
significant difference in the numbers of quantified proteins identified
from these two cell lines indicates that our newly developed strategy
is indeed applicable for selective labelling and enrichment of newly
synthesized proteomes in cell-specific manner. The high-confidence
hits identified from labelled A431 cells were found to be distributed
across various cellular components (Figure 3e), which is in
accordance with our previous profiling and imaging results.13
+
EGFR-free Cells
Figure 2. Selective labelling of nascent polypeptides in heterogeneous
environments, in (a) CHO-K1 cells transfected with EGFR-mCherry, and
(b) A431/CHO-K1 co-cultures. Cells were treated with Cetuximab-Tz (10
μg/mL, where applicable) for 30 min then TCO-PO (3 μM) for 24 h. Cells
were fixed with cold methanol and IF by using anti-puromycin antibody.
Total cells were detected by DAPI (blue). Scale bar = 25 μm.
that, in EGFR-expressing mammalian cells, Cetuximab-Tz binds to the
cell surface-bound EGFR and removes TCO group on TCO-PO to
generate free PO, which can be subsequently incorporated into
nascent polypeptides during ribosomal protein synthesis.
Importantly, this sequential incubation with Cetuximab-Tz and TCO-
PO is inert to cells that have no or low endogenous EGFR expression
levels.
In principle, in a heterogeneous cell environment, the unmasked
PO from this antibody-assisted strategy might diffuse extracellularly
to nearby cells lacking the target protein (e.g. EGFR), thus potentially
confounding cell-specific labelling. To determine whether this was
the case, we carried out PO labelling in heterogeneous cell cultures
with two different model systems (Figure 2): 1) CHO-K1 cells
transfected with EGFR-mCherry; and 2) co-culture of A431 and CHO-
K1 cells. As shown in Figure 2a, transient transfection of CHO-K1 cells
with an EGFR-mCherry plasmid led to the creation of a mixed culture
of cells with those EGFR-expressing cells indicated by the mCherry
fluorescence. Since the mCherry protein was fused to the C-terminus
of EGFR, it did not hinder the antibody binding site (Figure S11);
Cetuximab-Tz was found to selectively bind to the surface of
transfected CHO-K1 cells with fluorescent signals colocalized to those
from mCherry. Sequential treatments with Cetuximab-Tz and TCO-
PO followed by IF resulted in the labelling of nascent polypeptides
only in successfully transfected cells (Figure 2a; arrowed in red).
Importantly, no labelling of nascent polypeptides was detected in
cells not transfected with EGFR (arrowed in blue). We next
investigated whether the above conclusion also holds true for co-
culture of A431 and CHO-K1 cells. A431 cells were pre-stained with
Mitotracker before being mixed with an equal number of unstained
In conclusion, we have developed an antibody-assisted
strategy for cell type-specific labelling of newly synthesized
proteomes. Compared to existing strategies for cell-specific
proteomic studies, our approach provides the following
advantages: (1) this strategy is highly versatile and can be built
upon the vast array of available monoclonal antibodies that
This journal is © The Royal Society of Chemistry 20xx
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a)
Journal Name
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DOI: 10.1039/C7CC04536K
TCO-AY
Cetuximab-Tz
+
+
+
-
c)
250
130
100
70
55
35
25
Membrane
Cytoplasm
Cytoskeleton
Nucleus
A431 vs. CHO-K1 Hit Analysis
e)
d)
250
β-tubulin
Secreted
b)
200
150
100
50
Cetuximab-Tz+TCO-AY
TCO-AYONLY
ER
Mitochondrion
Cell projection
Cell junction
Lipid droplet
Lysosome
1)
2 )
3)
A431
EGFR(+)
Endosome
Golgi apparatus
0
0
15
30
35
A431
CHO-K1
Number of Proteins
Figure 3. Antibody-assisted incorporation of AY for mapping newly synthesized proteomes from A431 cells. a) TCO-AY structure. (right) Incorporation of
TCO-AY in nascent polypeptides. Cells were treated with Cetuximab-Tz (10 μg/mL, where applicable) for 30 min then TCO-AY (3 μM) for 24 h. Labelled
proteins were “click” with TER-azide, followed by SDS-PAGE and in-gel fluorescence scanning.13 β-tubulin was used as the loading control. b) Confocal
images of A431 cells treated with Cetuximab-Tz and TCO-AY. Labelled proteins were visualized by click chemistry with TER-azide (red). (blue): DAPI. Scale
bar = 15 μm. c) Scheme showing TCO-AY incorporation in newly synthesized proteomes by the antibody-assisted strategy to identify labelled proteins by
quantitative MS-based proteomics (SILAC). d) Graphical comparison of high-confidence proteins enriched in treated A431 and CHO-K1 cells. More than
1200 proteins were identified from A431 cells, of which 222 were considered high-confidence hits (plotted). e) Subcellular distribution analysis of high-
confidence proteins identified from treated A431 cells.
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require minimal optimization; (2) the system does not require
genetic modification and Is thus more suitable for tissue and
organism studies; (3) in future, other similarly “caged”
metabolites in addition to puromycin may be delivered to
targeted cells, to achieve cell type-specific labelling of other
biomolecules.
We gratefully acknowledge financial support from National
Medical Research Council (CBRG/0038/2013), Ministry of
Education (MOE2013-T2-1-048) of the Singapore government
and National Science Foundation of Zhejiang Province
(LQ16B020003).
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