Activity-Based Proteome Profiling Probes Based on Woodward's Reagent K with Distinct Target Selectivity

Article abstract of DOI:10.1002/anie.201602666

Woodward's reagent K (WRK) is a reactive heterocyclic compound that has been employed in protein chemistry to covalently and unspecifically label proteins at nucleophilic amino acids, notably at histidine and cysteine. We have developed a panel of WRK-der

Full text of DOI:10.1002/anie.201602666

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International Edition: DOI: 10.1002/anie.201602666
German Edition: DOI: 10.1002/ange.201602666
Activity-Based Probes Hot Paper
Activity-Based Proteome Profiling Probes Based on Woodwardꢀs
Reagent K with Distinct Target Selectivity
Yong Qian, Marc Schürmann, Petra Janning, Christian Hedberg,* and Herbert Waldmann*
Abstract: Woodwardꢀs reagent K (WRK) is a reactive hetero-
cyclic compound that has been employed in protein chemistry
to covalently and unspecifically label proteins at nucleophilic
amino acids, notably at histidine and cysteine. We have
developed a panel of WRK-derived activity-based probes and
show that surprisingly and unexpectedly, these probes are fairly
selective for a few proteins in the human proteome. The WRK-
derived probes show unique reactivity towards the catalytic
N-terminal proline in the macrophage migration inhibitory
factor (MIF) and can be used to label and, if equipped with
a fluorophore, to image MIF activities in living cells.
proline and can be employed to label and, if equipped with
a fluorophore, to image MIF activities in living cells.
The probes that were synthesized for the affinity enrich-
ment of proteins contained the reactive isoxazolium warhead
of WRK and a terminal alkyne handle interconnected via an
aromatic moiety and a spacer (Figure 1A). The analytical
workflow consists of the following steps (Figure 1C): 1) Pro-
teins are labeled with the ABPP probe, 2) an azide–biotin
[
3]
reporter tag (e.g., TAMRA–Biotin–N3) is added in a [3+2]
cycloaddition, 3) affinity enrichment using a streptavidin solid
support is performed, and 4) detection is done by 4a) SDS-
PAGE/in-gel fluorescence scanning and/or 4b) tryptic digest
and nano-HPLC MS/MS analysis. The probes P1–P10 (Fig-
ure 1D; for their complete structures, see the Supporting
Information, Figure S1) and a negative control probe (NP)
were synthesized by analogy according to established meth-
ods (see the Supporting Information).
I
n activity-based proteome profiling (ABPP), probes with
balanced, and often attenuated, chemical reactivity are
employed to covalently bind, isolate, and identify proteins
with enzymatic activity based on their mechanisms of action.
Typically, a nucleophile in the enzyme active site reacts with
an electrophile embedded in a selective probe. The final
selectivity profile of a probe is often a combination of the
relative electrophilicity of the binding site and the identity of
the scaffold. A variety of different electrophiles have success-
The WRK probes P1–P3, with terminal alkyne linkers
attached to different positions of the aromatic moiety (ortho,
meta, or para), were incubated at different concentrations
with HeLa cell lysate. Analysis of the resulting labeled
proteome using in-gel fluorescence detection (see the Sup-
porting Information for experimental details) revealed
a major fluorescent band at 15 kDa (Figure 2A, left panel),
with an intensity that depended on the concentration of the
probe and the substitution pattern of the aromatic ring in P1–
P3. The influence of the probe substitution pattern was
further ascertained with the isoxazolium salts P4–P6 (Fig-
ure 2A; for the structures, see Figures 1D and S1), and the
impact of the N substituent was determined by employing the
probes P7–P10 (Figure 2A; for the structures, see Figures 1D
and S1) with an alkyne PEG handle. The ortho-substituted
probes P5 and P6 showed a lower labeling efficiency than P4,
suggesting that ortho modification decreased the interaction
between probe and protein. P8, which contains a meta-
substituted alkyne PEG handle and an N-ethyl isoxazolium
core, showed the highest reactivity. Whereas in the presence
of an N-butyl group (P9), the reactivity was comparable, the
introduction of a methyl group (P7) or a cyclohexanemethyl
group (P10) attenuated the reactivity (Figure S2A). The
labeling reaction was very fast, and a cell lysate could be
efficiently labeled with P8 even within 1 min incubation
[
1]
fully been employed in ABPP probes.
[
2a]
Woodwardꢀs reagent K (WRK; Figure 1A) is a classical
electrophile employed in protein chemistry to covalently bind
to nucleophilic amino acids, notably cysteine and histidi-
[2b–d]
ne.
Mechanistically, WRK has been suggested to undergo
a base-induced ring-opening reaction, forming a ketenimine
intermediate, which subsequently traps a nucleophile (Fig-
[2a,b,e]
ure 1B).
WRK or analogues thereof have thus far not
been explored as ABPP probes of the human proteome,
although WRK has been reported to irreversibly inactivate
[
2e]
a number of enzymes in vitro.
Herein, we describe that
ABPP probes based on WRK are surprisingly selective
reagents that target the macrophage migration inhibitory
factor (MIF) by covalently modifying its catalytic N-terminal
[
*] Dr. Y. Qian, Dr. M. Schürmann, Dr. P. Janning, Prof. Dr. H. Waldmann
Max Planck Institute of Molecular Physiology
Otto-Hahn-Strasse 11, Dortmund (Germany)
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
Prof. Dr. C. Hedberg
Department of Chemistry, Chemical Biology Centre (KBC)
Umeå University, 90187 Umeå (Sweden)
E-mail: christian.hedberg@umu.se
(Figure S2B). Labeling was strongly diminished after heat
denaturation, suggesting that the labeling reaction needs the
intact proteome (Figure S2C). Interestingly, the probes could
be used over a wide pH range (pH 4.0–9.0; Figure S3).
P2- and P8-bound proteins were identified by proteomic
analysis after probe ligation, pull down, and in-gel as well as
Prof. Dr. H. Waldmann
Technical University Dortmund
Department of Chemistry and Chemical Biology
Otto-Hahn-Strasse 6, Dortmund (Germany)
[4]
on-bead tryptic digestion. Table 1 shows the hit frequencies
of proteins after in-gel digestion of the 15 kDa band for
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201602666.
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MIF was also enriched in
control experiments with cell
lysates from HeLa, HEK293,
and MCF-7 cell lysates when
P8 was used, whereas it was
not detected when DMSO or
the negative probe NP served
as the control (Figure S4).
Pre-incubation of HeLa
cell lysate with WRK (5/
2
1
5 equiv,
5 min) prior to the addition
100 mm/500 mm,
of 20 mm P8 efficiently inhib-
ited the labeling of MIF after
pull down (Figure 2B), thus
suggesting that WRK com-
petes with P8 for the same
binding site in the same
target. P8 labeling of MIF
can also be prevented with
the WRK derivatives PA and
PBr (Figure 2C; for the
structures, see Figures 1D
and S1), and pretreatment
with the covalent MIF inhib-
[
5]
itor 4-IPP (Figure 2C) fully
blocked the labeling of MIF
with P8. In contrast, P8-la-
beled proteins remained
unchanged after treatment
with the non-covalent MIF
inhibitor ISO-1 (Figure 2C).
MIF is a widely expressed
cytokine that plays crucial
physiological
roles
in
immune and inflammatory
[
6]
responses.
Upon stimula-
tion, MIF is released and
further promotes the produc-
tion of inflammatory media-
[7]
tors, such as TNF and IL-1.
An elevated level of MIF is
associated with inflammatory
and autoimmune disease and
[
6,8]
tumor growth.
Thus MIF
has the potential to serve as
a biomarker and therapeutic
Figure 1. Design of WRK probes. A) General structure of the WRK probes. B) Base-induced reaction between
WRK and nucleophiles as suggested by Woodward. C) Detection and identification of probe-labeled
proteome by the ABPP strategy. D) Chemical structures of probes P1–P10 (see the Supporting Information
for the syntheses and the complete structures). TBTA=tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine,
TCEP=tris(2-carboxyethyl)phosphine.
[9]
target. MIF is a keto–enol
[5,10]
tautomerase,
and can be
inhibited with either the com-
[11]
petitive inhibitor ISO-1 or
with the covalent inhibitor
4
-IPP, which directly targets
[5]
compound P2. MIF was the only protein identified as a hit in
four independent replicates, which was further validated by
Western blot analysis after pull down (Figure 2B). Affinity
enrichment experiments using P2 or P8 as baits and on-bead
digestion showed that the probes bind several other proteins
as well, but most of them to a lower extent (Tables S1 and S2).
the catalytically active N-terminal proline residue. Further-
more, isothiocyanates, as well as aminophen metabolites, have
also been reported to inhibit MIF.
[12]
Analysis of the WRK derivatives in a WST-1 proliferation
assay indicates that the WRK probe is only weakly cytotoxic
even at 100 mm (Figure S5). Cell permeability was confirmed
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Figure 2. In vitro reactivity of WRK probes. A) Concentration-dependent in vitro labeling with representative WRK probes in HeLa cell lysates
10 min, 258C, pH 7.4), tagged with the TAMRA azide using click chemistry, and separated by SDS-PAGE. In-gel fluorescence scanning
(
TM
experiments were performed with the ChemDoc MP system (Bio-Rad). Fluorescent gel images shown in gray. MW=molecular weight.
B) Confirmation of the major fluorescent band as MIF by Western blot analysis against a specific anti-MIF antibody, analysis after pull down on
streptavidin beads and after competition with WRK. C) In vitro competitive labeling of MIF with P8; HeLa cell lysates were pre-incubated with
different inhibitors (100 mm of the MIF inhibitors 4-IPP and ISO-1 or the WRK derivatives PA and PBr; 10 min) and then incubated with 10 mm P8
for another 10 min; analysis by Western blot after pull down. D) Structures of the probes NP, P2, and P8, the WRK derivatives PA and PBr, and
À1
the MIF inhibitors 4-IPP and ISO-1. E) Binding site of PBr. MIF (5 mL, 0.7 mgmL ) was incubated with 100 mm PBr in PBS buffer (pH 7.4) for
1
0 min at 258C. After acetone precipitation, MIF was digested with GluC and Trypsin. The resulting peptides were detected by nanoHPLC-MS/MS
and analyzed with MaxQuant to identify the modification sites. Left: MS/MS spectrum of m/z=770.85. Most peaks could be assigned to the full
y ion series of the N-terminal peptide of MIF (Pro1 to Arg11) carrying the probe PBr bound to proline 1 (Pro1). Right: Enlarged region of the MS/
MS spectrum for the fragment of proline 1 bound to the probe PBr (b ion). The simulated isotope pattern of this fragment is shown below.
1
by the incubation of HeLa cells with Rhodamine-tagged P8
Figure 3A) and co-localization of P8 with MIF by two-color
immunofluorescence analysis of these cells with a fluores-
cently labeled anti-MIF antibody (Figures 3A and S6). The
labeling efficacy in intact cells depends on the probe structure
in the same manner as shown for the lysate experiments
inhibitors prior to P8 incubation (4-IPP, PBr; Figure S9).
These observations suggest that the observed TAMRA
fluorescence signal reflects the selective targeting of MIF by
P8 even in intact cells.
(
Based on these findings, we synthesized a probe suitable
for the direct imaging of MIF in cells. The fluorescent probe
TP (Figure 3E) consists of a WRK reactive group bound to
(
Figures S7–S9), and the immunofluorescence staining inten-
[13]
sity for probes P8 and P2 behaves accordingly (Figure S6).
Labeling was abolished upon treatment of cells with MIF
a naphthalene ring (see the Supporting Information for its
synthesis). Consequently, it could be reactive towards nucle-
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Table 1: Hit frequency of proteins after four independent affinity
enrichments and in-gel tryptic digestion of the 15 kDa band using
compound P2 as the bait.
After treatment of HeLa cells with 100 mm TP for two hours,
confocal fluorescence microscopy imaging in the probe
[
a]
channel (l = 405 nm) showed a strong fluorescence signal
ex
Protein
Gene
MIF
M [kDa]
Frequency
4
for TP treated cells, but not for cells treated with the
corresponding negative control probe (NTP; Figure 3B). To
investigate whether TP can detect the known upregulation of
w
Macrophage migration
inhibitory factor
Thioredoxin
Galectin-1
Protein S100-A16
Protein S100-A4
12.476
[14]
TXN
11.737
14.716
11.801
11.728
3
3
3
3
MIF by lipopolysaccharides (LPS),
we pretreated HeLa
À1
LGALS1
S100A16
S100A4
cells with 1 mgmL LPS for 24 hours. After TP addition,
LPS-treated cells showed a much stronger fluorescence signal
than the control (Figure 3C). We transfected HeLa cells with
MIF siRNA to silence the MIF gene. Here, a significant
decrease in the blue fluorescence signals compared with the
siRNA control group was observed (Figure 3C), thus indicat-
ing that TP may serve as an imaging probe to detect the up/
downregulation of endogenous MIF in living cells.
[
a] Proteins identified as hits in at least three out of four experiments are
listed. Hit definition: The protein is identified with at least two unique
peptides in the compound sample after affinity enrichment, the overall
intensity of the peak areas of the peptides belonging to the protein is
8
greater than 1”10 , and the intensity of the protein after affinity
enrichment using the compound bait is at least ten times higher than the
intensity of the same protein in the corresponding negative control (or
the protein is not identified in the control).
To enable the live-cell imaging and subsequent in situ
profiling of endogenous MIF by gel-based ABPP, a dual-
purpose activity-based probe (TPP; for its structure, see
Figure 3E) was developed. This probe is based on the same
structure as TP and contains the same alkyne handle as
structures P7 to P10. All control experiments yielded similar
ophilic residues in the active site of MIF. TP shows bright
fluorescence and low cytotoxicity (Figures S10 and S11).
Figure 3. A) Immunofluorescence staining of HeLa cells with P8. HeLa cells were treated with P8 (10 mm) or DMSO for 2 h at 378C. Then, the
cells were fixed, permeabilized, and tagged with the Rhodamine110 azide using click chemistry; MIF was stained with a specific anti-MIF antibody.
Two-color immunofluorescence analysis showing the significant colocalization of P8-tagged Rhodamine110 with MIF. Scale bar: 50 mm.
B) Confocal fluorescence microscopy imaging of living HeLa cells with TP and the negative-control probe NTP (100 mm, 2 h). C) Images of HeLa
À1
cells with TP (100 mm, 2 h) with or without LPS (1 mgmL , 24 h pretreatment), or the universal scrambled negative controls SiRNA or SiRNA MIF
(
(
100 nm, 48 h pretreatment). One-photon image (l =405 nm) for the TP probe. Scale bar: 10 mm. D) Images of HeLa cells with TPP or NTPP
ex
100 mm, 2 h, 378C). One-photon images (l =405 nm) for the various probes; TPP was tagged with the Rhodamine110 azide using click
ex
chemistry; MIF immunofluorescence staining with a specific anti-MIF primary antibody and an Alexa Fluor 594 conjugated secondary antibody.
Merged image of the three channel images. Scale bar: 25 mm. E) Chemical structures of the probes NTP, TP, NTPP, and TPP.
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results as described for the structures mentioned above: Acknowledgements
1
) Concentration-dependent labeling showed that 500 nm
TPP was sufficient for acquiring a significant fluorescence
signal (Figure S12), 2) the enrichment of MIF was confirmed
by immunoblot analysis after labeling of cell lysate and intact
cells with TPP after pull down (Figures S13 and S14),
We thank the Max-Planck Society for generous support. Y.Q.
is grateful to the Alexander von Humboldt Foundation for
a postdoctoral fellowship. C.H. is grateful for generous
support from the Knut and Alice Wallenberg Foundation
(Sweden).
3
) pretreatment of cell lysate with 100 mm TP, PA, PBr,
-IPP, or WRK completely abolished the labeling by TPP
4
(
Figure S15), and 4) pretreatment of the HeLa cells with
Keywords: activity-based probes · fluorescent imaging ·
inhibitors (100 mm 4-IPP or TP for 2 h) significantly decreased
the labeling of MIF by TPP (Figure S14).
To confirm the suitability of TPP for live-cell imaging,
HeLa cells were incubated with TPP and imaged in the probe
protein labeling · proteomics
How to cite: Angew. Chem. Int. Ed. 2016, 55, 7766–7771
Angew. Chem. 2016, 128, 7897–7902
channel (l = 405 nm) by confocal fluorescence microscopy,
ex
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Received: March 16, 2016
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3102 – 13105.
Published online: May 9, 2016
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