Using peptidyl aldehydes in activity-based proteomics

Article abstract of DOI:10.1016/j.bmcl.2009.04.148

The broad inhibitory spectrum of aldehydes and the possibility that amino acid residues modulate their specificity point to the potential of using peptidyl aldehydes as activity-based probes. Here, we establish the potential of peptidyl aldehydes in activity-based proteomics by synthesizing different probes and using them to specifically label a well-known serine protease in an activity-dependent manner. From our results, peptidyl aldehydes emerge as promising activity-based probes that enable multiple enzymatic-class detection by substrate recognition and can be used in diverse activity-based proteomics applications like protein identification and activity profiling.

Full text of DOI:10.1016/j.bmcl.2009.04.148

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3752–3755
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Using peptidyl aldehydes in activity-based proteomics
Eduard Sabidó a,b, Teresa Tarragó b, Ernest Giralt a,b,
*
a Universitat de Barcelona, Departament de Química Orgànica, Martí Franquès 1–11, 08028 Barcelona, Spain
b Institut de Recerca Biomèdica, Baldiri Reixach 10–12, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The broad inhibitory spectrum of aldehydes and the possibility that amino acid residues modulate their
specificity point to the potential of using peptidyl aldehydes as activity-based probes. Here, we establish
the potential of peptidyl aldehydes in activity-based proteomics by synthesizing different probes and
using them to specifically label a well-known serine protease in an activity-dependent manner. From
our results, peptidyl aldehydes emerge as promising activity-based probes that enable multiple enzy-
matic-class detection by substrate recognition and can be used in diverse activity-based proteomics
applications like protein identification and activity profiling.
Received 13 February 2009
Revised 29 April 2009
Accepted 30 April 2009
Available online 6 May 2009
Keywords:
Aldehydes
Peptides
Ó 2009 Elsevier Ltd. All rights reserved.
Activity-based proteomics
Prolyl oligopeptidase
Activity-based proteomics (ABP) is a chemical strategy that uses
probes that covalently bind an enzyme active site for protein activ-
ity profiling and for discovering new therapeutic targets and en-
zyme inhibitors. Natural substrates and covalent inhibitors of
diverse enzymatic classes (e.g., proteases,1–6 glycosidases,7,8 and
phosphatases9) have been used for designing several activity-
based probes. In most cases, the reactive moiety of these probes
consists in electrophilic functional groups such as fluorophospho-
nates,1,2 epoxides,3,5 and acyloxymethylketones.10,11
the potential of using peptidyl aldehydes as activity-based
probes. Here, we explore for the first time the potential of pepti-
dyl aldehydes in ABP. For this purpose, we synthesized different
probes and, as a proof of principal, we used them to specifically
label a well-known serine protease in an activity-dependent
manner.
Prolyl oligopeptidase (POP; EC 3.4.21.26) is a post-proline ser-
ine protease that hydrolyses small proline-containing peptides.28
POP is involved in the regulation of many bioactive peptides
in vivo like substance P and thyrotropin-releasing hormone, among
others29–32 and has been associated to several neuropsychiatric
disorders like schizophrenia and bipolar affective disorder.33
Although the mechanism of action of this protease remains un-
known, several studies suggested that POP may produce its effect
through the metabolism of inositol-1,4,5-triphosphate, a key mol-
ecule in the transduction cascade of neuropeptide signaling.34,35 In
our laboratory, POP was recently cloned from human brain RNA,
expressed in Escherichia coli, and an homologous model based on
the X-ray structure of porcine POP was obtained.36
Aldehydes are electrophilic functional groups that have inter-
esting inhibitory properties toward different kinds of proteolytic
enzymes. Many compounds with an aldehyde moiety have been
recently described as covalent reversible inhibitors of serine and
cysteine proteases like trypsin, thrombin,12 and cathepsins13–15
among others.16–19 Apart from these proteases, aldehydes also in-
hibit metalloproteases20 and aspartic proteases21,22 like
c-secre-
tase, HIV protease, and renin, and they can also interact with
some non-proteolytic proteins like tyrosine phosphatases23,24 and
SH2 domains.25
Although aldehydes can inhibit a wide number of enzyme types
due to their broad reactivity, several studies succeeded in limiting
their interaction to certain enzymatic subclasses.13,18,26,27 Obtain-
ing specific aldehyde inhibitors has been possible with the syn-
thesis of peptidyl aldehydes and the subsequent modification of
the amino acid residues placed in positions P1 and P2 according
to the natural substrates of each enzyme.26,27
To evaluate the use of peptidyl aldehydes in ABP, three peptidyl
aldehyde activity-based probes, Aha-Bpa-Pro-Pro-H (1), Aha-Bpa-
Ahx-Pro-Pro-H (2), Aha-Bpa-Peg-Pro-Pro-H (3) (Aha: hexynoic
acid; Ahx:
e-aminohexanoic acid; Bpa: benzoylphenylalanine;
Peg: 15-amino-4,7,10,13-tetraoxapentadecanoic acid) were syn-
thesized and used to specifically label active POP (Fig. 1a). It is
worth mentioning that unlike phosphonates, sulfonates and other
reactive groups traditionally used in activity-based probes, the use
of aldehydes facilitates probe synthesis through easy and rapid so-
lid-phase peptide synthesis (SPPS) strategies.
The broad inhibitory spectrum of aldehydes and the possibil-
ity that amino acid residues modulate their specificity point to
Peptidyl aldehyde probes were initially designed from the well-
* Corresponding author.
E-mail address: ernest.giralt@irbbarcelona.org (E. Giralt).
known covalent POP inhibitor, Z-prolyl-prolinal (ZPP, Fig. 1b),37
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.148

E. Sabidó et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3752–3755
3753
included an alkyne functional group in the N-terminus to allow
fluorescent and biotin label conjugation by Cu(I)-catalyzed Huis-
gen [3+2] cycloaddition.43,44
O
a
Peptidyl aldehyde probes 1–3 were synthesized by SPPS using a
semicarbazide resin45,46 and dipeptides Fmoc-Bpa-Pro-OH, Fmoc-
Ahx-Pro-OH, and Fmoc-Peg-Pro-OH were used during peptide
elongation to avoid the formation of diketopiperazines.47 In all
O
O
1
N
N
H
N
H
O
O
O
O
cases L-amino acids were used. Recognition of POP active site by
H
N
probes 1–3 was confirmed by competitive inhibitory assays with
a well-known covalent probe, fluorophosphonate rhodamine (FP-
Rh), which binds to the active site of serine hydrolases.48 In these
assays POP was incubated for 15 min with increasing amounts of
N
N
H
2
3
N
H
O
O
O
either probe or ZPP. Afterwards, FP-Rh (1 lM) was added and the
O
incubation was maintained for an additional 60 min. After resolv-
ing the reaction on an SDS–PAGE, the FP-Rh-labeled POP was visu-
alized using a fluorescence scanner. All three compounds 1–3
showed excellent inhibitory properties toward POP (Fig. 1c) cor-
roborating that the introduction of additional bulky residues like
benzophenone was not drastically affecting their ability to recog-
nize the active site of the target protein.
O
H
N
O
N
N
H
N
H
4
O
O
O
O
O
Once peptidyl aldehyde probes were synthesized and their
inhibitory properties were confirmed, next assays focused on ver-
ifying if these compounds were proper activity-based probes, that
is, if they could label POP in an activity-dependent manner and dis-
tinguish its active form from heat-denatured samples. To confirm
this point, active and heat-denatured POP were incubated with
b
c
O
N
N
H
O
O
O
5 μM
25 μM
100 μM
probes 1–3 at 1 and 25 lM for 15 min at rt and crosslinked by
UV light irradiation for an additional 60 min at 4 °C. Afterwards,
samples were labeled using a trifunctional tag that incorporates
both a biotin and a rhodamine moiety49 (TriN3, Supplementary
Fig. 1), and they were analyzed by SDS–PAGE and visualized with
a fluorescence scanner. These assays showed that the probe speci-
ficity could be lost at high concentrations possibly due to the broad
aldehyde reactivity and to the photocrosslinking step. However,
peptidyl aldehydes are satisfactory activity-based probes when
used at low concentrations (Fig. 2a). These results were further
confirmed with pull-down experiments where the crosslinked
probe–protein complex was labeled with the TriN3 tag, fished out
of the sample using avidin beads and detected by Western Blot
ZPP + - -
+ - - + - -
- - + - - +
1
- - +
ZPP + - -
+ - - + - -
- - + - - +
2
- - +
ZPP + - -
+ - - + - -
- - + - - +
3
- - +
using either an in-house
was performed with probe 3 (1
tive form of POP was fished out from the sample (Fig. 2b).
a
-POP antibody or streptavidin. This assay
M), and in both cases only the ac-
Figure 1. (a) Structure of peptidyl aldehyde probes Aha-Bpa-Pro-Pro-H (1), Aha-
Bpa-Ahx-Pro-Pro-H (2), Aha-Bpa-Peg-Pro-Pro-H (3). Probes were synthesized by
SPPS using a t-Bu/Fmoc standard strategy except for the first amino acid residue,
which was coupled in the semicarbazide resin as Fmoc-Pro-H in THF and acetic acid
(1%). (b) Structure of Z-prolyl-prolinal. (c) Competitive inhibitory assays to evaluate
the recognition of POP active site by the new synthesized probes. In these assays
l
Finally, activity-based probe 3 was also used for direct mass
spectrometry protein identification using an nLC–MS/MS ap-
proach. In this case, probe–protein complexes were also labeled
with the TriN3 tag, fished out with avidin, but analyzed by re-
verse-phase chromatography followed by MS/MS identification.
MS/MS data were analyzed with the SEQUEST software using the
IPI database. The maximum false positive rate was set to 1% with
PeptideProphet and ProteinProphet. This data analysis led to the
identification of POP in active samples and not in heat-denatured
controls (Fig. 2c). These results show that peptidyl aldehydes are
adequate activity-based probes not only for specific in-gel activity
profiling of proteases but also for other ABP applications like pro-
tease identification using mass spectrometry.
To verify the behavior of peptidyl aldehyde probes in complex
environments, further experiments were performed mixing mouse
brain homogenates and recombinant POP. First, incubation and
photocrosslinking times were optimized (Supplementary Fig. 2a)
and the established conditions were used to identify the most
sensitive probe among the synthesized compounds (Supplemen-
tary Fig. 2b). Probe 3 was selected for subsequent experiments in
which brain homogenates were mixed with increasing amounts
of recombinant POP and incubated for 15 min with the probe
followed by UV light irradiation during an additional 60 min.
Although endogenous POP could not be detected by in-gel analysis,
POP (20 ng) was incubated for 15 min at 25 °C with increasing amounts (5
25 M and 100 M) of either probe or ZPP. Afterwards, FP-Rh (1 M) was added
and the incubation was maintained for an additional 60 min at 25 °C. The assay was
lM,
l
l
l
resolved on an SDS–PAGE and the FP-Rh-labeled POP was visualized using
a
fluorescence scanner.
and subsequently modified to fulfill the requirements previously
described for ABP probes.38,39 One of these requirements is the
establishment of covalent irreversible bonds between the probe
and the target protein. However, aldehydes establish covalent
reversible bonds with the active site of serine and cysteine prote-
ases and non-covalent bonds when interacting with aspartic prote-
ases and metalloproteases. This aldehyde reversibility could
constitute a certain restriction in the use of peptidyl aldehydes in
ABP applications. Therefore, to ensure covalent irreversible binding
between the synthesized probes and the target protein a photore-
active benzophenone residue was introduced at different positions
to induce photocrosslinking by UV light irradiation. This strategy is
similar to that previously used in the characterization of aspartic
proteases and metalloproteases with hydroxyethylenes40,41 and
hydroxamates.42 Finally, synthesized peptidyl aldehyde probes

3754
E. Sabidó et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3752–3755
25
50
125
250
C+
hPOP (ng)
1
2
3
a
Δ
- + - - + - - + - - + - -
-
Δ - Δ -
Δ
- - + - - + - - + - - + - ZPP
1 μM
MW (kDa)
25 μM
97
66
POP
-
Δ
b
c
42
α-POP
36
28
Streptavidin
Figure 3. Activity-dependent labeling of POP in mouse brain homogenates and
recombinant POP mixtures to establish the detection limit of peptidyl aldehyde
probes. Brain homogenates (1 mg/ml) were mixed with increasing amounts of
recombinant POP and incubated with activity-based probe 3 (1 lM). After UV light
irradiation samples were labeled with TriN3 and analyzed by SDS–PAGE and
DATABASE ID
PROTEIN
SAMPLE
IPI00220327.3
IPI00019359.3
IPI00009865.2
IPI00008164.1
IPI00747355.1
IPI00021304.1
IPI00217963.3
IPI00384444.5
IPI00290078.5
Keratin, type II cytoskeletal 1
Keratin, type I cytoskeletal 9
Keratin, type I cytoskeletal 10
Prolyl endopeptidase
Prolyl endopeptidase cDNA
Keratin, type II cytoskeletal 2 epidermal
Keratin, type I cytoskeletal 16
Keratin, type I cytoskeletal 14
Keratin 4
visualized with a fluorescence scanner. Pure recombinant POP labeled by FP-Rh
(1
denatured samples (
100 M) were also performed.
l
M) was used as a positive control (C+). Negative controls corresponding to heat-
D
) and to samples with a highly specific POP inhibitor (ZPP,
l
that peptidyl aldehydes emerge as promising activity-based probes
that enable multiple enzymatic-class detection by substrate recog-
nition and can be used in diverse ABP applications like protein
identification and activity profiling. Unlike other probes that only
target one specific type at a time (e.g., serine proteases), peptidyl
aldehydes can simultaneously target a variety of proteolytic en-
zymes. Therefore, they could become a valuable tool for identifying
unknown proteases by substrate recognition (e.g., those involved
in the activation of neuropeptide precursors), which could eventu-
ally lead to the establishment of new therapeutic targets. More-
over, the specificity resulting from the peptide sequence is an
important feature of peptidyl aldehyde probes that could also be
used to successfully monitor the activity of a particular subset of
proteins.
CONTROL
IPI00009865.2
IPI00220327.3
IPI00019359.3
IPI00021304.1
Keratin, type I cytoskeletal 10
Keratin, type II cytoskeletal 1
Keratin, type I cytoskeletal 9
Keratin, type II cytoskeletal 2 epidermal
Figure 2. (a) Activity-dependent labeling of POP at different probe concentrations.
In this experiment active (–) and heat-denatured ( ) POP (10 ng/ l) were incubated
with probes 1–3 at 1 and 25 M for 15 min at 25 °C and crosslinked by UV light
D
l
l
irradiation for an additional 60 min at 4 °C. Afterwards, samples were labeled using
the TriN3 tag and they were analyzed in an SDS–PAGE and visualized with a
fluorescence scanner. (b) Activity-dependent pull-down experiment. Probe
(1 M) was used for an activity-dependent labeling of POP (10 ng/ l). After
photocrosslinking and TriN3 incorporation, the crosslinked complex tag–probe–
protein was fished out of the sample using avidin beads and detected by Western
3
l
l
Blot (a-POP, streptavidin). (c) List of identified proteins by mass spectrometry (LC–
MS/MS) in samples with active POP and in heat-denatured controls after a pull-
down experiment. A Human IPI Database was used for protein identification.
Acknowledgments
This work was supported by MCYT-FEDER (Bio2008–00799 and
Generalitat de Catalunya (XRB and Grup Consolidat). E. Sabidó was
supported by a fellowship from Ministerio de Educación y Ciencia,
Spain.
these experiments made possible to set the lower limit of detection
of our probes around 25 ng of POP and, what is more important,
they validate the use of peptidyl aldehyde probes in complex mix-
tures (Fig. 3). In these experiments peptidyl aldehyde probes were
still able to distinguish active and non-active POP in complex
homogenates despite that the aldehyde reactive group could have
easily been masked or non-specifically inactivated by multiple
reagent species (e.g., primary amines). Moreover, peptidyl alde-
hyde probes remained quite specific and, other than POP, no other
proteins were clearly detected in an activity-dependent manner.
These observations confirm the importance of the peptidyl region
in limiting probe interaction to certain target proteases and make
peptidyl aldehyde probes useful for different ABP applications like
protease identification and substrate-limited activity profiling.
As shown in previous labeling assays, the photocrosslinking
step reduces probe selectivity and enforces the use of probes at
low concentration, which limits in-gel analysis sensitivity and
prevents the detection of endogenous POP. Obviously, peptidyl
aldehyde sensitivity needs to be improved for the general applica-
tion of these compounds as activity-based probes. However, the
sensitivity of peptidyl aldehydes reported here could still be
sufficient when working with high abundance proteins.
Supplementary data
Supplementary data (synthetic procedures, compound charac-
terization and protocols for activity-based assays and mass spec-
trometry) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2009.04.148.
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