A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags

Article abstract of DOI:10.1039/c6ob00810k

Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescenc

Full text of DOI:10.1039/c6ob00810k

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DOI: 10.1039/C6OB00810K.
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Systematic study of protein labeling by fluorogenic probes using
cysteine targeting vinyl sulfone-cyclooctyne tags
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a
c
a
d
a
B. Söveges, T. Imre, T. Szende, Á. L. Póti, G. B. Cserép, T. Hegedűs, P. Kele and K.
DOI: 10.1039/x0xx00000x
a
Németh *
www.rsc.org/
Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins
of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct
fluorescent derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is
carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys
targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free
strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE,
capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent
accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast
and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signal and fulfills all
requirements of bioorthogonality.
iodoacetamides are applied for chemical ligation in Cys
labeling schemes. In spite of the fact that vinyl sulfones show
superior performance compared to maleimides or
Introduction
Fluorescent labeling is undoubtedly one of the most
popular techniques for monitoring biological structures and
iodoacetamides vinyl sulfone based reagents are rarely
12, 13
1
,
2
applied.
They show excellent reactivities towards thiols
processes.
Side chains of some naturally occurring
with nearly quantitative yields without the formation of by-
products. Furthermore both the reagents and the products are
stable and have good water-solubility. In the last decade
bioorthogonal modulation schemes emerged as excellent
proteinogenic amino acids or either termini of proteins offer
suitable platforms for targeting. More specific, site selective
fluorescent tagging can be carried out on rare amino acids like
cysteine and tyrosine.
efficiently modulated via their thiol or phenolic hydroxyl group
3, 4
5-10
Such amino acid side-chains are
14-17
means for fluorescent labeling of proteins.
These
3
,
11
bioorthogonal reagents should suit the following main
requirements: (1) the kinetic, thermodynamic, and metabolic
stability of the reactants and products, (2) non-toxicity for
living organisms, (3) high selectivity, specificity and efficiency
under physiological conditions (ambient temperature and
pressure, neutral pH, aqueous conditions) in other words
either of the partners should exclusively react with each other
without any interference with other surrounding functions
present in live cells.
using specific reagents.
Generally, maleimides or
a.
Research Centre for Natural Sciences of Hungarian Academy of Sciences, Institute
of Organic Chemistry, Chemical Biology Research Group; H-1117 Budapest,
Magyar tudósok krt. 2. Hungary. Tel.: +36 1 382 6659; E-mail address:
nemeth.krisztina@ttk.mta.hu
Research Centre for Natural Sciences of Hungarian Academy of Sciences, Institute
of Organic Chemistry, MS Metabolomics Research Group; H-1117 Budapest,
Magyar tudósok krt. 2.
Research Centre for Natural Sciences of Hungarian Academy of Sciences, Institute
of Enzymology, Protein Research Group; H-1117 Budapest, Magyar tudósok krt. 2.
Hungary.
MTA-SE Molecular Biophysics Research Group, Department of Biophysics and
Radiation Biology, Semmelweis University; Tuzolto u. 37-47, H-1094 Budapest,
Hungary.
b.
c.
d.
Lately, we demonstrated the development of Cys specific
vinyl sulfone linkers bearing terminal alkyne as well as the
efficiency of a sequentially driven two-step modification
method of peptides using a fluorogenic dye in comparison with
Electronic Supplementary Information (ESI) available: Direct and sequential
fluorescent labeling of other proteins of interest are demonstrated. Detailed
syntheses routes and characterization data of molecules are available.
See DOI: 10.1039/x0xx00000x
10
one step direct labeling schemes (Fig. 1). These proof-of-
concept experiments underlined the advantages of
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sequentially driven modification schemes as these furnished
only one single fluorescent species i.e. the labeled peptide
providing the possibility for using no-wash conditions.
Results and discussion
Direct labeling of p38 with fluorescent linker-dye module
View Article Online
DOI: 10.1039/C6OB00810K
As a continuation of this work we aimed at further
The recombinant intracellular protein, p38 /MAPK14

investigating the potential of such vinyl sulfone based linkers (called p38 in the followings) (44 kDa; pI 5.6), a member of
and extending the applicability of these chemical reporters to mitogen-activated protein kinase family was selected as model
protein labeling schemes. However, proteins represent more protein, which is a key regulator element in signal transduction
22-24
challenging scaffolds as their rigid conformation compared to pathways.
peptides, and their cysteine residues are often hardly groups, two of which are surface-accessible (Fig. 2A).
accessible. In this work we intended to optimize the conditions
When p38 was treated with a cysteine-specific fluorescent
of the derivatization scheme in terms of time-scale analysis of linker-dye conjugate (LTA
More importantly, it contains four reduced thiol
10
D : Fig. 2C) an intensive fluorescent
1
the reaction and the number of tagged cysteines. signal evolution was observed by capillary zone
Furthermore, we made efforts to administer some novel linker electrophoresis with laser induced fluorescent detection (CZE-
molecules in order to eliminate the requirement for the toxic LIF) that reached maximum value within 90 minutes (Fig. 2B
Cu(I) catalyst in the click reaction (copper-catalyzed azide- and C). Labeling was found to be specific and covalent as LTA
D
1
alkyne cycloaddition (CuAAC)) between the linker and dye. The with pre-alkylated p38 gave only a very small peak in the
use of copper as a catalyst is often compromised in CuAAC electropherogram compared to untreated p38 (Fig. 2B). The
schemes due to the presence of chelating moieties in proteins disadvantage of this labeling scheme is that the unreacted
1
8
(
e.g. His-tagged) . To this end, we synthesized novel vinyl fluorescent reagent contributes to the overall signal depending
sulfone linkers bearing cyclooctyne moieties, which enables on the efficiency of the separation from the excess of LTA
the azide-alkyne reaction to proceed via strain-promoted
systematic tandem mass-spectrometric survey was
D .
1
A
1
1
azide-alkyne cycloaddition (SPAAC). We have tested two carried out involving trypsin digested plain p38 and LTA
treated p38 with or without iodoacetamide (IAM)
methyl 4-(cyclooct-2-yn-1-ylmethyl)benzoate) (2.4x10 M s pretreatment. IDA (Information Dependent Analysis) LC-MS
and a synthetically more challenging, but fast reacting experiments proved that the labeling was specific to thiol
COMBO (2-methoxycarbonyl-5,6,9,10-tetrahydro-7,8-dehydro- residues. Moreover it was also revealed that the Cys 119
benzocyclooctene) (0.8 M s ) that was developed in our residue was the primary subject of labeling and Cys 162 being
D
1
1
9
cyclooctyne motifs, a slower reacting, easily accessible CyO,
-3
-1
-
(
1
17
)
-1 -1
2
0, 21
laboratory.
Labeling schemes were compared using the secondary target of LTAD as the amount of these free thiol
1
fluorescent vinyl sulfone reagents enabling direct labeling and containing peptide fragments decreased by 97 ± 2 % and 60 ±
sequentially applicable non-fluorescent linkers in combination 15 %, respectively (n = 4) in LC-MS/MRM (Multiple Reaction
with fluorogenic dyes.
Monitoring) measurements (Fig. 3). Accordingly, the average
number of bound LTA per p38 protein was 1.57 based on the
MS results being in good agreement with labeling efficiency
1.46 ± 0.18 (n = 5)) estimated from the ratio of absorbancies
D
1
(
at 420 nm and 280 nm, monitored by UV-Vis
spectrophotometry (ESI).
Apart from these surface accessible thiol groups,
derivatization of the remaining Cys residues together with
other amino acids were insignificant. These findings are in
good agreement with the X-ray structure of p38 (PDB ID:
2
2
1
A9U), which suggest similar order of the thiols (accessible
2
2
2
2
surface area (ASA): 91 Å , 23 Å , 2 Å and 1 Å for Cys 119, 162,
2
11 and 39, respectively).
Furthermore, in case of a mutant p38 (p38
C162S
) where one
25
of the cysteines was replaced by serine the intensity of
labeling signal was comparable (ASA of Cys 119: 103 Å ) to that
2
of the wild type protein as established by CE-LIF and native
polyacrylamide gel electrophoresis (CN-PAGE) (Fig. 2B; Fig. 5).
In another instance, similarly intensive and specific signal
could be generated on another key signaling protein, i.e. ERK2
(extracellular signal-regulated kinase 2), when treated with
L D (Fig. 2, Fig. 4). ERK2 is very similar to p38 regarding its
TA
1
size and pI (44 kDa, pI 6.3) but the former has seven free thiol
groups, four in the inner region, two on the surface and one in
26,
the active center of the protein (see ASA values in Table S1).
27
Fig. 1
Comparison of direct and sequential fluorescent modification
schemes.
2
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2
2
Fig. 2
A: Structure of p38 with free thiol groups (Protein Data Bank ID: 1A9U). B: Capillary zone electrophoresis with laser induced fluorescent detection
C162S
of fluorescent tagging of p38 (1, 2), p38
corresponds to signal of LTA
1
p38 followed by the increasing area of peak ‘b’ and decreasing area of peak ‘a’ in CZE-LIF. Insert: Structure of the fluorescent tag LTAD .
1
(3, 4) and ERK2 (5, 6) with LTAD ; [proteins with (2, 4, 6) or without (1, 3, 5) alkylation pretreatment]. Peak ‘a’
1
D and peak ‘b’ to the labeled protein. For details see Experimental section. C: Time-scale analysis of the derivatization process of
Though p38 was labeled efficiently and selectively, it
should be mentioned that labeling schemes of other proteins
used as models in the preliminary phase of this study brought
our attention to a series of limitations (see ESI). More
particularly, it was observed that during Cys-specific labeling,
some proteins (e.g. albumin and α1-acid glycoprotein) showed
strong ligand binding features where the undesirable non-
covalent labeling contributed to the overall fluorescent signal.
Furthermore, the accessibility of the Cys residues can further
limit the efficiency of the fluorescent derivatization of native
proteins.
Fig. 3 Representative MRM chromatograms of tryptic digests of p38 (1), Sequential labeling of p38 with new linkers and fluorogenic azide
alkylated p38 labeled with LCOMBO (2) p38 labeled with LCOMBO (3) and p38 dyes
1
labeled with LTAD (4). Peaks were assigned by their MRM transitions. Peaks
There are two options to circumvent the obstacle of the
false positive labeling due to the non-covalent interactions in
case of proteins, which have ligand binding capacity. One of
the resolutions would be the separation of the ligand-bound
linker-dye construct – which is a very time-consuming process
whilst the protein can degrade. There is a more elegant
are numbered by their elution order: a: peptide fragment [119-121]; b:
internal standard – peptide fragment [46-49]; c: peptide fragment [150-
1
65]; d: peptide fragment [24-45]; e: peptide fragment [190-220]; f: peptide
fragment [119-121] conjugated with LCOMBO; g: peptide fragment [150-165]
conjugated with LCOMBO; h: peptide fragment [119-121] conjugated with
TA 1 1
L D ; i: peptide fragment [150-165] conjugated with LTAD .
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solution, however, i.e. to apply fluorogenic dye(s) in sequential
labeling route. The main advantage of driving the labeling
reaction in a sequential manner using fluorogenic dyes is that
the dyes applied are not fluorescent until unreacted even if
View Article Online
DOI: 10.1039/C6OB00810K
28-32
adsorbed non-specifically at biological surfaces
therefore
provides no-wash procedures. To this end we developed
bifunctional linkers (LCOMBO, LCyO) with vinyl sulfone warhead
and cylooctyne moieties as biorthogonal functional groups (Fig
4
.; see details of the synthesis in the Experimental Section
Scheme 1). These strained rings allow copper free SPAAC
reactions with fluorogenic azide dyes.
Fig. 4 Structures of the bifunctional linkers
Testing of fluorogenic azide dyes. Our objective was to Table 1 The evolution of fluorescent signal of dyes (D
1 6
-D ) incubated with
elaborate the performance of our cyclooctynylated vinyl
LCyO or LCOMBO and changes of intensity of fluorescent signal in the presence
sulfone linkers in combination with fluorogenic dyes in Cys- of reducing agents after two hours is shown.
specific labeling schemes. Firstly, we have reacted our new
10, 28, 29, 32
linkers and six fluorogenic azide dyes (D -D )
(Table
Fluorogenic azide dyes
1
6
1
). As expected, the substantial difference between the
[a]
1
[a]
2
[a]
3
[b]
4
[c]
5
[a]
D
D
D
D
D
D
6
19, 20
reactivity of the two cyclooctyne platforms
resulted in less
intense fluorescence increase in case of LCyO than for LCOMBO
Table 1) therefore we decided to use LCOMBO for further
studies.
It is crucial that the fluorogenic dyes are stable under the
(
labeling conditions or their decomposition product is non-
fluorescent. To get a deeper insight into the stability of our
10, 28, 29, 32
fluorogenic azides
in reducing media, we have
conducted a systematic screening study in order to assess the
most suitable fluorogenic dyes. The fluorogenic performance
of azide-quenched systems can be compromised in many
ways. It is known, that azides are prone to reduction in the
presence of strong reducing agents (ME, TCEP). However,
0
Changes in fluorescence intensity (I/I )
L
CyO
0.9
1.5
7.3
1.6
2.3
2.2
2.4
1.1
1.4
1.4
2.0
2.5
3.1
11.2
0.8
1.1
1.5
1.6
2.6
1.5
18.9
1.4
8.0
1.2
1.0
0.9
0.9
4.0
2.4
10.3
1.5
1.4
1.2
1.2
7.0
0.9
3.2
L
COMBO
33
TCEP
ME
1.6
there are also instances in the literature that cysteine itself
–
even in peptides and proteins – can reduce azides (Table 1).
Furthermore, it should be kept in mind that cytoplasm
represents a highly reductive medium (cf. glutathione (GSH))
where the application of azide functions (or other
bioorthogonal chemical reporters e.g. unnatural amino acids)
needs careful attention as loss of azide integrity can lead to
the appearance of non-specific signal (Table 1). In order to
check the reducing effects of proteins containing reduced Cys,
our azides D -D were mixed with native albumin (BSA) –
2.0
Cys
2.6
GSH
BSA
16.2
52.2
as the greatest increase that corresponds to the highest
reactivity with LCOMBO and LCyO and the highest fluorogenicity.
Accordingly, dyes D5 and D4 and D1 showed the highest
stability and the largest reactivity. The fluorescence generated
upon reaction with LCOMBO was slighter for D than D , which is
1
6
having a reduced cysteine in position 34. Evolution of intense
fluorescent signals was observed in case of D , D D and D in
1
2,
3
6
the presence of native BSA while no fluorescence could be
detected using prealkylated form of the protein. These results
indicated that fluorescent decomposition products generated
upon the reaction with the reduced Cys and those compounds
associated non-covalently to the protein giving a false positive
signal of selective labeling. On the contrary, in the case of D4
4
5
in good agreement with quantum yield data published
previously. Consequently, we have selected D5 for further
29
applications. In addition, in order to compare results obtained
with L D we used dye D as well.
TA
1
1
Conjugation of linkers to the protein. We have applied the
same reaction protocol for treatment of p38 with
cyclooctynylated linker LCOMBO as with L D . The specificity of
and D only slight or insignificant emission was detected in the
5
presence of reductive BSA in comparison with control
experiments using non-reducing prealkylated BSA (Table 1,
Table S2) demonstrating that these dyes remained intact.
The most appropriate dyes were selected from the series
of D -D based on their stability and reactivity. We ranked the
TA
1
LCOMBO binding to the protein was confirmed by MS (Fig. 3). As
expected, the two surface accessible Cys (119 and 162) were
targeted at similar modification ratios as observed with L D1
95 ± 3 % and 68 ± 5 %, respectively, n = 3). Furthermore, we
checked by subsequent addition of LTAD1 to cyclooctyne-
treated p38 for any residual accessible thiol groups on p38. To
TA
1
6
(
dyes based on the changes in fluorescence intensity values i.e.
we preferred the smallest increase in the presence of reducing
agents – including BSA – referring to the best stability as well
4
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our delight, no substantial fluorescent signal could be detected The conjugation process followed a normal satVuierwatAiroticnle Otynlpinee
for these p38 samples on SDS-PAGE suggesting that pattern with a rapid increase in fluorescDeOnIc: e10i.1n0t3e9n/sCi6tyOBa0t0h81ig0hK
derivatization with the linker was complete within two hours reactant concentrations that reached a plateau after 30
(
Fig. S2).
minutes.
Noteworthy and a pleasant finding is the fact, that
Labeling of linker conjugated p38 with fluorogenic azide
dyes. We have successfully conducted sequential labeling fluorescence intensity of LCOMBO-D5 adduct was substantially
schemes using LCOMBO and D or D monitored by CZE-LIF and less intensive compared to p38-LCOMBO-D conjugate since the
1
5
5
C162S
CN-PAGE (Fig. 5, Fig. S3). p38
and ERK2 gave similar results fluorescent intensity of D increases 20 and 100 times upon
5
upon tagging with LCOMBO and D1 or D5 (Fig. 5). It is worth tagging with LCOMBO and with p38-LCOMBO conjugate,
mentioning that the emission wavelength of conjugated D1 respectively.
was found to be strongly dependent on the structure of the
linker used but indifferent of the protein applied.
Conclusions
Specificity of the tagging was tested on pre-alkylated p38.
Covalent modifications were identified exclusively at
accessible cysteine residues as demonstrated by MS
experiments. Only slight non-covalent binding was
experienced in case of native p38 by CZE-LIF and CN-PAGE
Herein we have demonstrated that cycloocytynylated thiol-
specific vinyl sulfone warhead bearing chemical reporter(s) are
suitable to derivatize appropriate protein platforms. The
bioconjugation reaction of the native proteins is fast and
selective. Subsequent click reaction with carefully selected
fluorogenic dyes ultimately provides the generation of an
intensive fluorescence signal and fulfills all requirements of
bioorthogonality. Therefore, we propose the application of a
new vinyl sulfone and COMBO functionalized thiol specific
linker for the installation of a fast reacting bioorthogonal
cyclooctyne motif. The COMBO moiety can be efficiently
targeted with carefully selected fluorogenic dyes to achieve
intensive and specific fluorescence.
(Figure S3) indicating the existence of ligand binding effects,
similarly to albumin (Fig. S4), although to a much less extent.
Time scale analysis showed (Fig. 6) that the click reaction is
fast and completed within 20 min in case of mixing LCOMBO with
D5, and slightly slower for the protein-LCOMBO conjugate with
D .
5
Experimental
Organic syntheses
C162S
Fig. 5 Fluorescent tagging of p38, p38
1
and ERK2 with LTAD , and with
Unless otherwise indicated, all starting materials were
L
COMBO and dyes D
1
and D
fluorescent emission (λex = 365 nm) and lower to the Coomassie staining.
Samples 1: native, untreated p38 2: p38 labeled with LTA
conjugated with LCOMBO and labeled with D
; 4: p38 conjugated with LCOMBO synthetized according to literature procedures.
5
monitored by CN-PAGE. Upper gel photo refers to obtained from commercial suppliers (Sigma-Aldrich, Fluka,
Merck, Alfa Aesar, Reanal, Molar Chemicals, Fluorochem) and
; 3: p38 used without further purification. CyO and COMBO were
D
1
1
9, 20
1
L D was
TA 1
C162S
C162S
10
and labeled with D
5
; 5: native, untreated p38
6: p38
labeled with prepared as described previously.
C162S
C162S
L
TA
1
D ; 7: p38
conjugated with LCOMBO and labeled with D
1
; 8: p38
Analytical thin-layer chromatography (TLC) was performed
conjugated with LCOMBO and labeled with D
ERK2 labeled with LTA
; 12: ERK2 conjugated with LCOMBO and labeled with D
5
; 9: native, untreated ERK2; 10: on silica gel 60 F254 precoated aluminium TLC plates from
; 11: ERK2 conjugated with LCOMBO and labeled with Merck. Visualization of TLC samples was performed with a
254/365 nm UV lamp and/or KMnO stain. Column
D
1
D
1
5
.
4
chromatography was carried out with silica gel (0.06-0.2 mm)
from Zeochem.
NMR spectra were recorded on a Varian Inova 500 MHz
and Varian 600 MHz NMR spectrometers. Chemical shifts (δ)
are given in parts per million (ppm) using solvent signals as the
reference. Coupling constants (J) are reported in Hertz (Hz).
Splitting patterns are designated as s (singlet), d (doublet), t
(
(
triplet), qr (quadruplet), qv (quintuplet), m (multiplet), dd
doublet of a doublet), td (triplet doublet), dt (doublet triplet),
brs (broad singlet). The exact masses were determined with an
Agilent 6230 time-of-flight mass spectrometer. IR spectra were
obtained on a Bruker Alpha-P spectrometer on a single-
reflection diamond ATR unit.
Fig. 6 Time-scale analysis of the click reaction of LCOMBO (2) and p38-
COMBO (3) conjugate with D compared to D alone (1) monitored by
fluorescent imaging ( ex: 355 nm/ em: 450 nm; n = 3).
L
5
5


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Scheme 1 Synthesis of LCOMBO and LCyO: a) di-tert-butyl dicarbonate, DCM,
Calculation of solvent accessibility of Cys residues in p38.
View Article Online
DOI: 10.1039/C6OB00810K
t
2
5°C, 14 h, 67 %; b) cat. BuOK, DVS, THF, N
2
, 25°C, 45 min, 52 %; c) CH
COCl, Atoms other than the target proteins’ were removed from the
3
structures. The cleaned structure files were submitted to
2
MeOH, 0-25°C, 4 h, 98 %; d) HBTU, HOBt×H O, EDIPA, DMF, 25°C, 3 h, 20 %
accessible surface area (ASA) and relative ASA (RSA)
(LCOMBO) and 47 % (LCyO).
34
calculations using DSSP and the dssp Python package
[
http://jbloom.github.io/mapmuts/dssp_module.html] using
the 'Tien2013' scale of maximum accessible surface area of
3
5
amino acids.
Conjugation processes
Conjugation of model proteins and L D . 50 µM protein
TA
1
and 300 µM L D were mixed and incubated for 2 hours at
TA
1
room temperature in dark and in 50 mM sodium HEPES buffer
pH 8.0).
(
Conjugation of model proteins and linkers. 100 µM
protein and 1 mM linker (LCyO or LCOMBO) were mixed and
Syntheses of cyclooctynylated linkers (LCOMBO, LCyO). Boc- incubated for 2 hours at room temperature in dark and in 50
protected 2-(methylamino)-ethanol ( ) was subjected to a mM sodium HEPES buffer (pH 8.0). The linker excess was
Michael-type of addition with divinyl sulfone (DVS) in the removed by SpinPrep column (Sigma, St. Louis, MO, USA) filled
2
t
presence of catalytic BuOK to afford
Removal of the Boc protecting group was effected by Chemicals, Sweden).
treatment with trifluoroacetic (TFA) acid to furnish
3 in medium yield. with Sephadex G-25 “Fine” desalting gel (Pharmacia Fine
4
.
However, it was noted that the product was always slightly
Labeling of model proteins conjugated with linker with
fluorogenic azide dyes. 50 µM protein and 300 µM fluorogenic
or dyes were mixed and incubated for 2 hours at room
contaminated upon TFA mediated deprotection.
2
0
(
LCOMBO, L ) were prepared using COMBO-COOH (5)
CyO
1
9
CyO-COOH (
6
)
under standard peptide coupling conditions temperature in dark and in 50 mM sodium HEPES buffer (pH
(EDIPA, HOBt, HBTU – N,N-diisopropylethylamine, Thus, we 8.0).
applied in situ generated methanolic HCl solution, which
furnished analytically pure hydrochloride salt of the free amine
Alkylation of model proteins. The model proteins were
(4) in quantitative yield. Bifunctional chemical reporters alkylated with 700 times excess of iodoacetamide (IAM) for 1
hydroxybenzotriazole,
2-(1H-benzotriazol-1-yl)-1,1,3,3- hour at room temperature in dark and in 50 mM sodium
tetramethyluronium
hexafluorophosphate,
respectively) HEPES buffer (pH 8.0). The unreacted reagent was removed by
SpinPrep column (Sigma, St. Louis, MO, USA) filled with
Sephadex G-25 “Fine” desalting gel (Pharmacia Fine Chemicals,
(Scheme 1; for more details see ESI).
Fluorogenic dyes. Dyes D -D (Table 1) have been prepared Sweden).
1
6
1
0, 28, 29, 32
as described previously.
Stock solutions were
dissolved in 10 mM in DMSO.
Analytical methods
Model Proteins. Human serum albumin, bovine serum
albumin, carbonic anhydrase I (from human erythrocytes),
Capillary electrophoresis. Background electrolyte (BGE)
components Tris (Tris(hydroxymethyl)-aminomethane and
superoxide dismutase I (from bovine erythrocytes) and α -acid
1
Tris-HCl,
HEPES
(N-[2-hydroxyethyl]piperazine-N’-[2-
glycoprotein were purchased from Sigma (St. Louis, MO, USA).
All kinase protein constructs were recombinantly expressed in
Escherichia coli Rosetta (DE3) pLysS (Novagen) cells with
ethansulfonic acid]), were purchased from Sigma (St. Louis,
MO, USA) sodium hydroxide and phosphoric acid were
purchased from Merck GmbH (Darmstadt, Germany).
2
5
C162S
standard techniques. p38α, p38α
mutant and ERK2 were
Capillary electrophoresis was performed with an Agilent
expressed with N-terminal cleavable hexahistidine tag.
Recombinant proteins were purified with standard
chromatographic techniques. The procedure involved a Ni-
affinity chromatography step that was followed by an ion
exchange step on a Resource Q column (GE Healthcare). The
elution buffer contained 20 mM Tris (pH 8.0), 500 mM NaCl, 10
CE
Capillary Electrophoresis 3D system (Agilent Technologies,
Waldbronn, Germany) applying bare fused silica capillary
having a 64.5 cm total and 56 cm effective length with 50 μm
I.D. (Agilent Technologies, Santa Clara, CA, USA). On-line
absorption at 200 nm was monitored by DAD UV-Vis detector.
Laser (ex: 355 nm) (Teem Photonics, Meylan, France) induced
fluorescent (LIF) signal was observed by ZetaLIF Discovery
detector (Picometrics, Labege, France). The capillary was
thermostated at 25°C. Between measurements, the capillary
%
glycerol, and 2 mM TCEP. Concentrations of stock solutions
were 20 mg/mL (500 µM), 10 mg/mL (250 µM) and 13 mg/mL
C162S
(
325 µM) for p38, p38
as well as for ERK2, respectively.
6
| J. Name., 2012, 00, 1-3
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Page 7 of 8
Org aP nl ei ca s &e dB oi o nmo to al ed cj uu sl at rm Ca hr ge imn si stry
Journal Name
ARTICLE
was rinsed subsequently with 0.1 M HCl, 1.0 M NaOH, 0.1 M bottom, 384-well plate for fluorescence (Corning, Corning, NY,
View Article Online
DOI: 10.1039/C6OB00810K
NaOH and distilled water for 3 minutes each and with BGE for USA).
5
minutes. 500 mM Tris-HCl (pH 9.0) buffer was used for p38
samples and 200 mM sodium phosphate (pH 9.0) for the other
proteins. Samples were injected by 5×10 Pa pressure for 6 from our former publication. Briefly, 50 µL of p38 (2 mg/mL,
sec. Runs were performed in the positive-polarity mode with 50 µM, 2.5 nmol) with or without pretreatment and 10 μL 0.2
Mass Spectrometry. Tryptic digestion method was adapted
3
36
3
0 kV.
% (w/v) RapiGest SF (Waters, Milford, USA) solution buffered
Predicted acidic dissociation constant (pKa) of L D dye with 50 mM ammonium bicarbonate (NH HCO ) were mixed.
TA
1
4
3
and theoretical isoelectric point (pI) of the proteins were 1.5 µL of 45 mM DTT in 100 mM NH HCO were added and
4
3
calculated by ChemAxon’s Marvin Calculator Plugin software kept at 60°C for 30 min. After cooling the sample to room
version 5.9.2; URL: http://www.chemaxon.com/marvin) and temperature, 2 μL of 100 mM iodoacetamide in 100 mM
by ExPASy Bioinformatics Resorce Portal NH HCO were added and placed in the dark for 30 min. The
URL:http://web.expasy.org/compute_pi/), respectively.
(
4
3
(
reduced and alkylated protein was then digested by 5 μL (2
mg/mL) trypsin (the enzyme-to protein ratio was 1 : 10)
Clear native polyacrylamide gelelectrophoresis. Samples (Sigma, St. Louis, MO, USA). The sample was incubated at 37 °C
were diluted with sample buffer (62.5 mM Tris-HCl (pH 6.8) + for 60 min. To degrade the surfactant, 7 μL of formic acid (500
5 % glycerol + 0.01 % bromophenolblue) in 1:1 ratio. The size mM) solution was added to the digested p38 sample to have
2
of the native polyaclyamide gels were 8.5 cm×7.5 cm×0.1 cm. the final concentration between 30 and 50 mM (pH ≈ 2) and
They were the combination of 4 % concentration and 10 % was incubated at 37°C for 45 min. For LC-MS analysis, the acid
separation PAGE gels (acrylamide:bisacrylamide ratio was treated sample was centrifuged for 10 min at 13 000 rpm.
3
7.5:1; and gel buffer was 375 mM Tris-HCl (pH 8.8). 5.0 µg of
QTRAP 6500 triple quadruple – linear iontrap mass
protein was loaded per lane. Separations were carried out in spectrometer, equipped with a Turbo V Source in electrospray
Mini-Protean Tetra Cell (Bio-Rad, Hercules, CA, USA) using 25 mode (AB Sciex, CA, USA) and a Perkin Elmer Series 200 micro
mM Tris/192 mM Glycine; (pH 8.3) as running buffer and 150 V LC system (Massachusetts, USA) was used for LC-MS/MS
voltage for 70 min. Gels were documented by Fluorchem FC2 analysis. Data acquisition and processing were performed
Imager (Alpha Innotech, San Leandro, CA, USA) at ex: 365 nm. using Analyst software version 1.6.2 (AB Sciex Instruments, CA,
Furthermore, gels were stained for proteins with Coomassie- USA). Chromatographic separation was achieved by Vydac 218
Brillant-Blue.
TP52 Protein & Peptide C18 column (250 mm × 2.1 mm, 5µm).
Sample was eluted with a gradient of solvent A (0.1 % formic
UV-Vis spectroscopy. Calibrations for p38 and dye-LTAD1 acid in water) and solvent B (0.1 % formic acid in ACN). Flow
were obtained at 280 nm and 420 nm in quartz cuvette with 1 rate was set to 0.2 mL/min. The initial conditions for
mm light pathlength by Jasco 7800 UV-Vis spectrophotometer. separation were 5 % B for 4 min, followed by a linear gradient
Dye/protein ratio was estimated from A420/A280 with to 30 % B by 40 min, from 41 to 50 min 90 % B, from 51 to 55
corrections according to the
details see ESI).

values obtained (for more min 90 % B is retained; from 56 to 60 min back to initial
conditions with 5 % eluent B. The injection volume was 10 µL
(300 pmol on column). The MS/MS system was operated
Screening of reduction of fluorogenic azide dyes by under multiple reaction monitoring (MRM) mode, with a dwell
fluorescent detection. The effect of reducing agents TCEP, ME, time of 100 ms for each transition, using nitrogen as the
Cys (300 µM), glutathione (5 mM) and BSA (50 µM) – with or collision gas, set at “high”.
without prealkylation – on fluorogenic azide dyes (300 µM; D1-
D6) were studied based on the increase of their fluorescent
Acknowledgements
signals (ex: 355 nm/ em: 410 nm; ex: 355 nm/ em: 450 nm;
and ex: 410 nm/ em: 500 nm) by Biotek Synergy 2 Cytation 3
imaging plate reader with Gen5 software version 2.07 (Biotek
Winooski, VT, USA) in round bottom, 384-well plate for
fluorescence (Corning, Corning, NY, USA). In comparison, the
The authors thank Attila Reményi and Anita Alexa for the
conductive discussions. The authors would like to thank
András Herner for preparation of some of the applied
fluorogenic dyes. Ilona Kawka, Marianna Rakács and Holly
Sofka are greatly acknowledged for the technical support. The
research was financially supported by the Hungarian Academy
of Sciences and the Hungarian Scientific Research Fund
evolution of fluorescent signal of the dyes (D –D ) upon
1
6
conjugation with LCyO and LCOMBO after two hours of incubation
was determined.
(
Lendület Grant - LP 2013-55/2013 and OTKA-NN-110214,
Time-trace analysis with monitoring the fluorescent
signal. Derivatization reaction (20 µL) between the fluorogenic
OTKA-NN-116265).
azide dye (300 µM; D ) and the L
(50 µM) or p38 (50 µM)
COMBO
5
conjugated with LCOMBO were monitored based on the
generation of the fluorescent signal (ex: 355 nm; em: 450 nm)
by Biotek Synergy 2 Cytation 3 imaging plate reader with Gen5
software version 2.07 (Biotek Winooski, VT, USA) in round
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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ARTICLE
Journal Name
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P. Shieh, M. J. Hangauer and C. R. Bertozzi,ViJe.wAAmrtic.leCOhnelmine.
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