Europium-labeled activity-based probe through click chemistry: Absolute serine protease quantification using 153Eu isotope dilution ICP/MS

Article abstract of DOI:10.1002/anie.201108277

Click and analyze: The titled probe was synthesized by conjugating a sulfonyl fluoride and azido unit using click chemistry to give SF-Eu, which can react specifically with serine (Ser) in the active site of serine protease (SP). Combination of the method

Full text of DOI:10.1002/anie.201108277

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Angewandte
Communications
DOI: 10.1002/anie.201108277
Protein Quantification
Europium-Labeled Activity-Based Probe through Click Chemistry:
Absolute Serine Protease Quantification Using 153Eu Isotope Dilution
ICP/MS**
Xiaowen Yan, Yacui Luo, Zhubao Zhang, Zhaoxin Li, Qiang Luo, Limin Yang, Bo Zhang,
Haifeng Chen, Peiming Bai, and Qiuquan Wang*
The development of molecular mass spectrometry (MS),
including various types of molecular MS with electrospray
ionization (ESI) and matrix-assisted laser desorption/ioniza-
tion (MALDI), as well as isotope-labeling strategies have
greatly advanced relative protein quantification recently.[1]
Among the limited number of methods reported using the
molecular MS for absolute protein quantification, the results
are primarily achieved using multiple reaction monitoring
(MRM) of unique peptides representing target proteins on
a triple-quadrupole molecular MS.[2] The MRM strategy has
proven to be highly reproducible and accurate, but it calls for
proper and unique peptide standards to generate high-quality
MRM analysis. In some cases, the unique peptides from the
target proteins have low ionization efficiencies and are
difficult to analyze using the molecular MS. It is especially
difficult to synthesize hundreds and thousands of peptide
standards based on a whole proteome, but the standards are
necessary, because different peptides have varying ionization
efficiencies in the molecular MS. To overcome the intrinsic
limitations of the molecular MS, the feasibility of using
elemental mass spectrometry [especially inductively coupled
plasma mass spectrometry (ICP/MS)] was recently explored
for absolute protein quantification through the determination
of either the naturally occurring heteroelements (S,[3] P, [4]
Se[5]) of proteins and metals in the metalloproteins,[6] or the
exogenous elements (I,[7] Hg,[8] Ln[9]) attached to the protein
side chains. Besides extremely high sensitivity and selectivity
as well as a broad dynamic range of up to nine orders of
magnitude, a signal that is independent of chemical species
(peptides and/or proteins) is another unique advantage of
ICP/MS over the molecular MS when considering the
absolute protein quantification. On the basis of this advant-
age, proteins can be quantified absolutely by coupling high-
performance liquid chromatography (HPLC) with species-
unspecific isotope dilution ICP/MS without the use of
individual peptide or protein standards,[10] which are required
in the molecular MS. It should, however be pointed out that
a prerequisite for the success of the absolute protein
quantification is the peak purity during HPLC. In other
words, all the peptides and proteins in a complex proteome
sample should be baseline separated. Actually, this is nearly
impossible using the currently available separation tech-
niques when the absolute protein quantification is applied to
a real-world proteome sample. In contrast, recent targeted
proteomics for obtaining the information for targeted pro-
teins, rather all the proteins in a proteome, has developed
rapidly.[2] Based on this concept of targeted proteomics, we
considered using a biospecific labeling strategy, such as
activity-based protein profiling (ABPP),[11] for a class of
functional proteins to solve the problems mentioned above. In
contrast to the immuno affinity assay, whose specificity comes
from the noncovalent interaction between the antigen and
antibody,[12] ABPP is based on the design and synthesis of an
activity-based probe, which consists of two functional groups:
1) a group on an irreversible enzyme inhibitor that covalently
binds to the active site of an active enzyme, and 2) a reporter
group to readout the enzyme activity. By specifically labeling
a class of active enzymes in a complex proteome sample,
ABPP can decrease the sample complexity and reduce the
separation requirements of current techniques, especially
when using ICP/MS because only the labeled target enzymes
are determined.
[*] Dr. X. W. Yan, Y. Luo, Z. Zhang, Z. Li, Prof. L. M. Yang, Dr. B. Zhang,
Prof. Dr. Q. Q. Wang
Department of Chemistry and the Key Laboratory of Analytical
Sciences, College of Chemistry and Chemical Engineering
Xiamen University, Xiamen 361005 (China)
E-mail: qqwang@xmu.edu.cn
Prof. Dr. Q. Q. Wang
State Key Laboratory of Marine Environmental Science
Xiamen University, Xiamen 361005 (China)
Dr. Q. Luo, Dr. H. Chen
Institute for Biomedical Research
Xiamen University, Xiamen 361005 (China)
To the best of our knowledge, we report herein the first
absolute protein quantification method using species-unspe-
cific isotope dilution HPLC/ICP/MS together with an activity-
based labeling strategy (see Figure S1 in the Supporting
Information), which is expected to boost the development of
the absolute protein quantification in the near future. As one
of the largest and most widely distributed enzyme families in
human cells, serine hydrolase comprises about 1% of the
human proteome including about 176 serine proteases (SPs),
which hydrolyze proteins.[13] SPs were used as examples to
demonstrate the feasibility of this strategy.
Dr. P. Bai
Department of Urology, Xiamen University Affiliated Zhongshan
Hospital, Xiamen 361004 (China)
[**] This study was financially supported by the National Natural
Science Foundation of China (21035006) and the National Basic
Research 973 Program (2009CB421605). Prof. John Hodgkiss is
thanked for assistance with the English in this paper.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201108277.
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To design and synthesize an activity-based SP probe
containing a lanthanide (which is a very sensitive element in
ICP/MS and has a low background noise in biological
systems), we chose a widely used irreversible SP inhibitor,
4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF).[14] The
sulfonyl fluoride (SF) group in AEBSF can bind covalently
and specifically to the hydroxy group of the serine residue in
the active site of SPs, thus resulting in a sulfonyl–SP derivative
which is stable for long periods of time.[15] On the other side of
the AEBSF, the amine group is conjugated with 6-heptynoic
acid to produce an SF–alkyne unit (see Figure S2 in the
Supporting Information), which can then undergo click
(HATU), and an organic base, N,N-diisopropylethylamine
(DIPEA), were used to complete the reaction. The SF–alkyne
unit was synthesized and purified using HPLC, and the
structure was confirmed using ESI/MS (see Figure S3 in the
Supporting Information), and 1H and 13C NMR spectroscopy
(Figures S4 and S5).
To investigate the efficiency of labeling SPs with the
purified SF–alkyne, we used chymotrypsin and elastase as
model SPs. After incubating 1 mm SF–alkyne with freshly
prepared chymotrypsin and elastase in 100 mm Tris-HCl
(pH 7.5), the model SPs were completely labeled with one
SF–alkyne unit within 1 hour (even though the numbers of the
serine residues of chymotrypsin and elastase were 26 and 22,
respectively), as confirmed by using ESI/MS (Figure 1). The
deconvolution molecular weights (DMs) of intact chymo-
trypsin and elastase were 25443 Da and 25896 Da, respec-
tively; and the DMs of these two SPs increased to 25736 Da
and 26186 Da, respectively after they were labeled with one
SF–alkyne unit (MW= 311). Thus, the DMs match the
theoretical molecular weights (TMs) of 25734 Da and
26187 Da, respectively. Differences of 2 and 1 Da between
the DM and TM values are due to the relatively low-mass
resolution of the ion trap MS employed. These results
demonstrated that SF–alkyne was quite efficient and specific
for labeling the hydroxy group of the serine residue in the
active site of the SPs, and is consistent with other studies on
the labeling of chymotrypsin[17] and elastase[18] with SF
derivatives.
chemistry with
a
europium-loaded azido-monoamide-
DOTA (azido-DOTA-Eu; Scheme 1). The click chemistry is
an [3+2] cycloaddition reaction between a terminal alkyne
and azide using CuI as a catalyst.[16] In this proof-of-concept
study, we tried to attach azido-DOTA-Eu to SPs in two
different ways: 1) SPs were first conjugated with SF–alkyne
and subsequently labeled with azido-DOTA-Eu through
a click reaction (Scheme 1A); 2) The azido-DOTA-Eu unit
was conjugated to SF–alkyne to give SF–alkyne/azido-
DOTA-Eu (SF-Eu), which was then used to label the SPs
(Scheme 1B). In both cases, the SF–alkyne unit needs to be
synthesized first. To minimize the hydrolysis of the SF group
in alkaline aqueous solution, the amidation reaction between
ASBSF and 6-heptynoic acid was carried out in DMF, and
a
carboxyl activating agent, O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate
In the next step, we optimized the
click reaction between azido-DOTA-
Eu and SF–alkyne (see Figure S6a in
the Supporting Information), with
respect to the reducing reagents,
tris(benzyltriazolylmethyl)
amine
(TBTA; CuI-stabilizing ligand),[19]
the reaction time, and the kinds of
buffer used. We found that sodium
ascorbate was a much more efficient
reducing reagent than tris(2-carboxy-
ethyl)phosphine in our reaction
system (see Figures S6b,c). When
three times the amount of azido-
DOTA-Eu was reacted with SF–
alkyne in a mixture containing 5 mm
sodium ascorbate, 1 mm CuSO4,
0.1 mm TBTA, 100 mm Tris-HCl
(pH 7.5), and 20% tert-butyl alcohol
for 1 hour, more than 99% of the SF–
alkyne was labeled with azido-
DOTA-Eu to produce SF-Eu (see
Figures S6c,d). The object of using
20% tert-butyl alcohol was to prevent
precipitation of TBTA in pure aque-
ous solution. When we attempted to
conjugate the SPs to SF–alkyne to
obtain alkyne-SF-SP, which was then
Scheme 1. Schematic illustration of the strategies for labeling serine protease using a Eu-labeled
activity-based probe. A) First, the hydroxy group of the serine residue in the active site of serine
protease was labeled with SF-alkyne, and subsequently labeled with azido-DOTA-Eu by click
chemistry. B) SF-alkyne-azido-DOTA-Eu probe (SF-Eu) was synthesized first by conjugating SF-
alkyne and azido-DOTA-Eu by click chemistry, and then used to label SP directly.
reacted
with
azido-DOTA-Eu
through the click chemistry
(Scheme 1A), the SPs unfortunately
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Figure 1. ESI/IT/MS spectra of a) intact chymotrypsin, b) intact elastase, c) SF-alkyne-labeled chymotrypsin, d) SF-alkyne-labeled elastase, e) SF-
Eu-labeled chymotrypsin, and f) SF-Eu-labeled elastase. DM and TM in the MS spectra denote deconvolution and theoretical molecular weights,
respectively.
precipitated out. The speculated reason for this phenomenon
was the use of TBTA, according to a previous report.[20]
However, TBTA is a very important reagent for stabilizing
CuI, and plays a catalytic role in the click reaction.[19] No SF-
Eu was found after 1 hour in the case where neither CuI nor
TBTA were used. We therefore changed our strategy
(Scheme 1B) such that the SF-Eu probe was synthesized
first and then used to label the SPs directly. The purified SF-
Eu was used to label chymotrypsin and elastase in 100 mm
Tris-HCl (pH 7.5), and the ESI/MS spectra showed that both
of the SPs were completely labeled with one SF-Eu unit
(MW= 946) within 1 hour and without any precipitate
(Figures 1e,f). Although SF-Eu is larger than SF–alkyne by
one DOTA-Eu group, it could still label the model SPs
efficiently. The linker between the SF and DOTA-Eu groups
could place the DOTA-Eu group far enough away from SF to
avoid a steric effects.
only chymotrypsin and elastase reacted with SF-Eu under the
labeling conditions. As shown in Figure 2a, SF-Eu and SF-Eu-
labeled peptides or proteins in the sample were baseline
separated on a C18 column using HPLC with the optimized
gradient elution program and UV (214 nm) detection (see the
Supporting Information). The effluent from the HPLC was
subsequently mixed with the enriched 153Eu standard 153Eu-
Table 1: Ten model peptides and proteins used.
No.
Name[a]
Number of
serine residues
Molecular weight
[kDa]
1
2
3
4
5
6
7
8
9
10
vasopressin
oxytocin
RNase A
somatostatin
Cyt C
lysozyme
BSA
chymotrypsin
elastase
0
0
15
1
1.0
1.1
13.7
1.6
12.3
14.3
66.4
25.4
25.9
29.0
0
10
28
26
22
30
A sample containing three peptides (vasopressin, oxy-
tocin, and somatostatin) and five proteins [ribonuclease A
(RNase A), cytochrome C (Cyt C), lysozyme, bovine serum
albumin (BSA), and carbonic anhydrase], together with the
two SPs was chosen to investigate the specificity of SF-Eu to
SPs (Table 1). The results obtained (Figure 2) suggested that
carbonic anhydrase
[a] The SWISS-PROT numbers for the proteins are given in the
Supporting Information.
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trations of chymotrypsin and elastase in the mixed protein
solution, prepared from the crude chymotrypsin and elastase
solid powder obtained from Sigma–Aldrich, were determined
to be (17.8 Æ 0.5) mmolLÀ1 and (9.1 Æ 0.3) mmolLÀ1, respec-
tively, and corresponded to (90.3 Æ 2.5)% and (46.9 Æ 1.3)%
within the purity values provided by the producer. The
detection limits of this method for the SPs were calculated to
be 0.2 fmol based on the signal-to-noise ratio (3s criterion) of
Eu in the mass-flow chromatogram with a RSD lower than
3% (n = 5 at pmol level). These quantitative results demon-
strated that the sensitivity and precision of 153Eu species-
unspecific isotope dilution HPLC/ICP/MS on the absolute
quantification of the SPs were very high and accurate. It
should be mentioned here that fluorophore[21] and isotope-
coded tags[22] based on ABPP have been used to monitor the
activities of the SPs, whereas the absolute quantification of
the SPs remains unreported, because of the limitations of the
detection techniques used. Obviously, the Eu-labeled activity-
based probe together with species-unspecific isotope dilution
ICP/MS overcame the limitations and realized the absolute
quantification of the SPs.
To validate the accuracy of the 153Eu species-unspecific
isotope dilution HPLC/ICP/MS with the Eu-labeled activity-
based probe, we further applied species-specific isotope-
dilution HPLC/ICP/MS to measure the contents of chymo-
trypsin and elastase in the sample. Different from specific-
unspecific isotope dilution, species-specific isotope dilution
method could account for any peptide/protein loss or
contamination during the processes from sample preparation
and HPLC separation through detection, which was carried
out by spiking the 153Eu-labeled SP standards into the natural
Eu-labeled sample and analyzed using the HPLC/ICP-MS
(Figure S1). To obtain the SP standards, we purified SF-153Eu-
labeled chymotrypsin and elastase from the crude protein
samples using HPLC, and the purified SF-153Eu-labeled
chymotrypsin and elastase were further quantified using the
standard Bradford assay and ICP/MS (data were not shown
here). When a known amount of 153Eu-labeled chymotrypsin
and elastase were spiked into the natural Eu-labeled sample
for 153Eu species-specific isotope dilution HPLC/ICP/MS
analysis, the ratio detected (Rdetected) of 153Eu-labeled chymo-
trypsin and elastase versus natural Eu-labeled chymotrypsin
and elastase matched the theoretical Rtheory very well (see
Figure S7 in the Supporting Information), and indicated the
accuracy of 153Eu species-specific isotope dilution HPLC/ICP/
MS. The concentrations of chymotrypsin and elastase in the
mixed protein solution prepared from the crude chymotrypsin
and elastase solid powder obtained from Sigma–Aldrich were
determined to be (0.45 Æ 0.02) mmolLÀ1 and (0.23 Æ
0.01) mmolLÀ1 corresponding to (95.2 Æ 3.3)% and (50.3 Æ
1.8)% in the crude solid proteases powders. Provided that
these results could be considered “true” values, those
obtained using 153Eu species-unspecific isotope dilution
HPLC/ICP/MS were in good agreement with the true
values, and the recovery of chymotrypsin and elastase were
within (94.8 Æ 2.7)% and (93.1 Æ 2.6)% (n = 5), respectively.
To demonstrate the feasibility of 153Eu species-unspecific
isotope dilution HPLC/ICP/MS for the absolute quantifica-
tion of the SPs in biological samples, different concentrations
Figure 2. Typical chromatograms of the peptide/protein sample using
a) HPLC/UV (214 nm), b) HPLC/ICP-MS for monitoring 153Eu and
151Eu isotopes with species-unspecific isotope dilution, c) the Eu mass-
flow chromatogram of the SF-Eu-labeled peptide/protein transformed
from (b) according to the on-line isotope dilution equation (Equa-
tion S1). Peak Nos. in (a) denote the corresponding peptide/protein
listed in Table 1.
(NO3)3 through a three-way connector, and the signals of
153Eu and 151Eu isotopes in the mixed effluent were monitored
using ICP/MS (see Figure S1). The data in Figure 2b indicates
that almost only chymotrypsin and elastase were labeled with
SF-Eu and detected using ICP/MS, whereas the ICP/MS
signals of the other peptides and proteins such as Cyt C and
lysozyme were below 1.0% compared with the signal of
chymotrypsin, thus demonstrating the specificity of SF-Eu for
the SPs. Moreover, it should be pointed out that even though
there were other peptides/proteins of the same retention time
as those of the labeled SPs on the HPLC, only the labeled SPs
would be determined when using ICP/MS.
The chromatograms for the 153Eu and 151Eu isotopes
(Figure 2b) show that the signal intensity of Eu decreased
when the acetonitrile content of the effluent for the C18
column was changed in accordance to the gradient elution
program (see the Supporting Information). To calibrate this
effect, as both isotopes are affected to the same extent and the
isotope ratio remains stable, the chromatograms for 151Eu and
153Eu isotopes were transformed into a Eu mass-flow chro-
matogram (Figure 2c) using the on-line isotope dilution
equation given in Equation S1 in the Supporting Information.
By integrating the peak areas corresponding to chymotrypsin
and elastase in the Eu mass-flow chromatogram, the concen-
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Purkayastha, P. Juhasz, S. Martin, M. Bartlet-Jones, F. He, A.
Jacobson, D. J. Pappin, Mol. Cell. Proteomics 2004, 3, 1154 –
1169.
of chymotrypsin and elastase were spiked into human serum
samples collected from Xiamen University Affiliated Zhong-
shan Hospital. We found the SPs could not be labeled and
detected when the concentration of the spiked chymotrypsin
and elastase was below 5 and 2.5 mm, respectively, mainly
because of the high level of SPs inhibitors such as alpha-
1 proteinase inhibitor (1–2 mgmLÀ1) in human serum.[23]
Within the ranges of 10–50 and 2.5–25 mm, the recoveries of
chymotrypsin and elastase, respectively, were found to be
from 34.0 to 51.0% and 18.4 to 56.1%, respectively (see
Figure S8 in the Supporting Information), which meant that
about 66.0 to 49.0% and 81.6 to 43.9% of these two SPs were
inhibited by the inhibitors in the human serum samples. This
phenomenon implied that one can additionally apply this
method to investigating the level of SPs inhibitors and their
degree of inhibition effect in a real biological sample, besides
the absolute quantification of the active SPs. We further
applied this strategy to quantify the active SPs in Tris-HCl
extractable fraction of rat tissues (kidney, heart, liver, lung,
brain and pancreas), and we found that different kinds of the
SPs were expressed in these tissues and they ranged from 0.4
to 7.6 nmolgÀ1 (see Figure S9). When 36 pmol chymotrypsin
and 12 pmol elastase were spiked, we found that the
recoveries of these two SPs in the tissues except pancreas
were above 90% (see Figure S10), which meant that the
contents of SPs inhibitors in the rat tissues were relatively low.
It should be noted that the recovery of chymotrypsin in
pancreas was only 38.4% while that of elastase was 96.7%,
thus suggesting that chymotrypsin-preferable inhibitors exist-
ing in the pancreas significantly inactivated chymotrypsin.[24]
All these results indicate the feasibility of this method for
analyzing the active SPs in real biological samples.
In conclusion, we developed a highly sensitive, precise,
and practical method for the absolute quantification of active
SPs. To the best of our knowledge, this is the first report of
using this newly designed and synthesized Eu-labeled activity-
based SP probe together with isotope dilution ICP/MS for
active SP specific labeling and absolute quantification.
Further application of this strategy is ongoing in our
laboratory to quantify other SPs. Not limited to the SPs
shown in this report, we believe that this method can be easily
extended to other classes of enzymes, such as cysteine
proteases,[11a,25] metalloproteases,[26] oxidoreductases,[27] and
cytochrome P450[28] by developing the corresponding lantha-
nide-labeled activity-based probes.
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Received: November 24, 2011
Revised: January 8, 2012
Published online: February 17, 2012
Keywords: europium · isotopic labeling ·
.
liquid chromatography · mass specrometry · proteins
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