A novel method for screening peptides that bind to proteins by using multiple fluorescent amino acids as fluorescent tags

Article abstract of DOI:10.1039/b920426a

We describe a new screening method for simultaneously detecting peptides that bind to a target protein by fluorescence obtained from fluorescent amino acid-modified peptides.

Full text of DOI:10.1039/b920426a

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A novel method for screening peptides that bind to proteins by using
multiple fluorescent amino acids as fluorescent tagsw
Mizuki Kitamatsu,* Midori Futami and Masahiko Sisido
Received (in Cambridge, UK) 30th September 2009, Accepted 16th November 2009
First published as an Advance Article on the web 30th November 2009
DOI: 10.1039/b920426a
We describe a new screening method for simultaneously detecting
peptides that bind to a target protein by fluorescence obtained
from fluorescent amino acid-modified peptides.
an acetyl group. These peptides were identified by MALDI-
TOF MS (Acd(KDD): calc. [M + H]+ = 1911.80, obs.
[M + H]+ = 1912.81; Cmr(DKD): calc. [M + H]+
=
1991.85, obs. [M + H]+ = 1992.89; Pyr(KEE): calc.
[M + H]+ = 1946.85, obs. [M + H]+ = 1947.71; Bad(KAD):
calc. [M + H]+ = 1917.83, obs. [M + H]+ = 1918.94;
A peptide library is generally used for screening peptides that
bind to a target protein. The phage display method and the
one-bead–one-compound method are widely used methods for
screening with a peptide library.1–5 These methods essentially
need to fix short peptides on very large carriers (sub-micron
order) such as phages and beads. This has a drawback because
the large carriers often bind to the target protein nonspecifically
and specific binding of peptides to the target protein is
inhibited. To overcome this drawback, we have developed a
new screening method for detecting peptides that bind to a
target protein. This new method does not need carriers.
Instead of carriers, multiple fluorescent amino acids are
connected to peptides. The fluorescent amino acids are utilized
as fluorescent tags in this method. Peptides that have bound to
a target protein are instantaneously identified and quantified
by fluorescence from the tags.
Ant(AAA): calc. [M + H]+ = 1749.78, obs. [M + H]+
=
1950.79; Edn(DDD): calc. [M + H]+ = 2011.75, obs.
[M + H]+ = 2012.69). A Cy3-labeled anti-FLAG M2 antibody
and non-labeled anti-FLAG M2 antibody used in this study
were purchased from Sigma-Aldrich (St Louis, MO, USA).
The peptides modified with a fluorescent tag and the
Cy3-labeled anti-FLAG M2 antibody were examined by
two-dimensional fluorescence (2D-FL) spectroscopy in
10 mM HEPES-NaOH buffer (pH 7.4, 150 mM NaCl)
containing 50% MeOH as shown in Fig. 2. The fluorescent
tags showed differences in maximum excitation/emission
wavelengths (Acd(KDD): 403/420 nm, Edn(DDD): 350/480 nm,
Bad(KAD): 485/512 nm, Cmr(DKD): 327/386 nm, Pyr(KEE):
344/378 nm, Ant(AAA): 359/408 nm, Cy3-labeled anti-FLAG
M2 antibody: 555/570 nm). We also examined the mixture of
these six peptides (each 10.0 nM, 30 pmol) and Cy3-labeled
anti-FLAG M2 antibody (0.2 nM, 0.6 pmol) by 2D-Fl
First, to examine whether a peptide that binds to a protein
specifically can be detected by our method, we used a
combination of a FLAG peptide (DYKDDDDK) and an
anti-FLAG antibody. It is known that a FLAG peptide
specifically binds to an anti-FLAG antibody.6 We prepared
a FLAG peptide and its mutant peptides modified with a
fluorescent amino acid as a fluorescent tag by conventional
solid-phase peptide synthesis (SPPS).7,8 Fluorescent amino
acids (Fmoc-Ala(Acd)-OH, Fmoc-Glu(Edn)-OH, Fmoc-
Ala(Bad)-OH, Fmoc-Lys(Cmr)-OH, Fmoc-Ala(Pyr)-OH,
and Fmoc-Ala(Ant)-OH) were purchased from Watanabe
Chemical (Hiroshima, Japan).9 Chemical structures of
peptides modified with a fluorescent tag and sequences of
these peptides are shown in Fig. 1. Fl indicates a fluorescent
amino acid. Chemical structures of fluorescent amino acids are
also shown in Fig. 1. To improve water solubility, two
glutamic acids are connected to the fluorescent amino acid.
Xn (n = 1–8) indicates a natural amino acid. The 8-mer
peptide consisting of Xn is a library moiety. A long spacer
consisting of ethylene glycol units (Sp6) connects the
fluorescent amino acid with the library moiety. Ac indicates
Department of Medical and Bioengineering, Graduate School of
Natural Science and Technology, Okayama University,
3-1-1 Tsushimanaka, Okayama 700-0082, Japan.
E-mail: kitamatu@cc.okayama-u.ac.jp; Fax: +81-86-251-8219;
Tel: +81-86-251-8219
w Electronic supplementary information (ESI) available: Synthesis of
new fluorescent amino acids and fluorescent tag-modified peptides. See
DOI: 10.1039/b920426a
Fig. 1 Chemical structure of the peptide modified with a fluorescent
tag and sequences of peptides. Chemical structures of fluorescent
amino acids.9
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Chem. Commun., 2010, 46, 761–763 | 761

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Fig. 3 2D-Fl spectrum of the fraction containing Cy3-labeled anti-
FLAG M2 antibody obtained after gel filtration chromatography of
the mixture of fluorescent tag-modified peptides and the Cy3-labeled
anti-FLAG M2 antibody.
Fig.
2
2D-Fl spectra of fluorescent tag-modified peptides, a
Cy3-labeled anti-FLAG M2 antibody and the mixture of these
peptides (each 10 nM) and the antibody (0.2 nM) over the wavelength
ranges of 300–600 nm for excitation and 305–650 nm for emission in
10 mM HEPES-NaOH (pH 7.4, 150 mM NaCl) containing 50%
MeOH at room temperature.
We preliminarily examined screening for EGFR-binding
peptides by this method. An EGFR (epidermal growth factor
receptor)12,13 is a cell-surface receptor, and mutations affecting
EGFR expression or activity can result in cancer. Therefore,
an EGFR-binding peptide is an attractive medical tool, and
some research groups screened for such a peptide by phase
display methods.14–17
spectroscopy (Fig. 2). Then concentrations of fluorescent
tag-modified peptides in the mixture were estimated by a
least-squares analysis of fluorescent intensity in the 2D-Fl
spectrum for the mixture on the basis of component spectra
(2D-FL spectra for the fluorescent tag-modified peptides).
Consequently, concentrations of these peptides in the mixture
were successfully quantified by this method (Bad(KAD): 11.6 nM,
Cmr(DKD): 10.8 nM, Pyr(KEE): 10.2 nM, Edn(DDD):
10.4 nM, Ant(AAA): 14.5 nM, Acd(KDD): 8.8 nM and
Cy3-labeled anti-FLAG M2 antibody: 0.2 nM).10 The results
indicate that fluorescence resonance energy transfer (FRET),
quenching and aggregation between peptides did not occur
under these conditions. The results also clarified that this
method can distinguish and quantify peptides in the mixture.
Next, the Cy3-labeled anti-FLAG M2 antibody (50 nM,
5.0 pmol), the non-labeled anti-FLAG M2 antibody (1 mM,
100.0 pmol) and the six peptides (each 1 mM, 100.0 pmol) were
mixed in 10 mM HEPES-NaOH buffer (pH 7.4, 150 mM
NaCl). After overnight incubation at 4 1C, the mixture was
fractionated by Superdex 75 pg size exclusion chromatography
(GE Healthcare, London, UK). A 2D-FL spectrum for the
fraction containing the Cy3-labeled anti-FLAG M2 antibody
was measured as shown in Fig. 3. Concentrations of all of the
components were determined by the protocol described above.
Acd(KDD) (FLAG peptide modified with Acd: 44.1 pmol) was
detected together with a nearly equimolar amount of the anti-
FLAG M2 antibody (54.0 pmol, estimated from fluorescence
intensity of Cy3). We estimated the dissociation constant (Kd)
of Acd(KDD)/anti-FLAG antibody complex by using least-
squares analysis of fluorescent intensity for fractions in which
the complex was observed. The Kd value was 17 Â 10À9 M,
which corresponds to a literature report of Kd (6.5 Â10À9 M).11
The somewhat higher value may be because of nonspecific
adsorption of these peptides and/or the protein to gel by using
gel filtration. Other peptides were not detected in the fraction
(Edn(DDD): 8.1 pmol, Bad(KAD): 5.7 pmol, Pyr(KEE):
0.6 pmol, Cmr(DKD): o0 pmol, Ant(AAA): o0 pmol). This
result indicates that the method can be utilized as a method for
detecting a peptide that binds to a protein specifically.
In this study, the extracellular domain of human EGFR
fused to the Fc region of human IgG via a linker peptide
(R & D Systems, Minneapolis, MN, USA) was used as a target
protein. Seven fluorescent tag-modified peptides were newly
synthesized by SPPS. Fluorescent amino acids (Fmoc-Lys-
(Moc)-OH, Fmoc-Lys(Mac)-OH, Fmoc-Lys(Tmr)-OH, Fmoc-
Orn(Cm3)-OH, Fmoc-Lys(Fam)-OH and Fmoc-Lys(Hoc)-OH)
were used as fluorescent tags on SPPS.18 Fmoc-Lys(Tmr)-OH
and Fmoc-Lys(Fam)-OH were purchased from ABD Bioquest
(Sunnyvale, CA, USA) and other fluorescent amino acids were
newly synthesized (see ESIw). Sequences of these peptides and
chemical structures of fluorescent amino acids are shown in
Fig. 4.
These peptides were examined by 2D-FL spectroscopy in
10 mM HEPES-NaOH (pH 7.4, 150 mM NaCl) containing
50% MeOH (maximum excitation/emission wavelengths:
348/400 nm for Moc(G/A/V), 377/464 nm for Mac(I/L/M),
401/421 nm for Acd(S/T), 411/451 nm for Hoc(D/E), 446/491 nm
for Cm3(N/Q/P), 501/524 nm for Fam(H/K/R), 542/569 nm
for Tmr(F/W/W)). Then these peptides were mixed with an
EGFR in 10 mM HEPES-NaOH buffer (pH 7.4, 150 mM
NaCl), and the mixture was incubated at 4 C for 2 h. Final
concentrations of the peptides were 190–280 mM (4.5–14 nmol)
and the final concentration of the EGFR was 3.9 mM
(190 pmol). The mixture was then fractionated by Superdex
Fig. 4 Sequences of the peptides modified with a fluorescent tag and
chemical structures of the fluorescent amino acids.18
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In summary, we have developed a new method for screening
peptides that bind to proteins. We synthesized 8-mer peptides
modified with a fluorescent amino acid. The peptides were
mixed with an anti-FLAG antibody and an EGFR before
incubation, and then the protein-binding peptides were recovered
by gel filtration chromatography. The binding peptides were
able to quantify simultaneously by this method. This method
combines fluorescence analysis with gel filtration and it also
will be able to combine the analysis with other separation
techniques (capillary electrophoresis, ultrafiltration and
affinity bead). Determination of an 8-mer peptide sequence
that binds to an EGFR by this method is currently underway.
Notes and references
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8 M. Kitamatsu, M. Shigeyasu, T. Okada and M. Sisido, Chem.
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9 Abbreviations used are as follows: Fmoc, 9-fluorenylmethoxy-
carbonyl; Acd, 2-[9(10H)-acridonyl]; Edn, 5-naphthyl sulfonic acid;
Cmr, 7-methoxycoumarin-4-yl; Bad, [benzo[b]acridin-12(5H)-on-
2-yl]; Pyr, 1-pyrenyl; Ant, 2-anthryl.
Fig. 5 Quantities of the fluorescent tag-modified peptides and the
EGFR estimated from 2D-FL spectra by least-squares analysis.
10 These calculated values were scattered in comparison with the
theoretical value (10 nM), especially the value for Ant (14.5 nM).
The scattering of the values seemed to be affected by the nature of
the fluorescence group. Choice of fluorescence group may be
important for analysis correctly by this method.
11 G. J. Wegner, H. J. Lee and R. M Corn, Anal. Chem., 2002, 74, 5161.
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18 Abbreviations used are as follows: Moc, 4-(7-methoxycoumaryl);
Mac, 7-dimethylaminocoumarin-4-yl; Tmr, 5(6)-tetramethyl-
rhodamine; Cm3, 3-coumarin 343; Fam, 5(6)-fluorescein; Hoc,
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75 pg size exclusion chromatography. These fractions were
measured by 2D-FL spectroscopy. Concentrations of all
components in each fraction were quantified by the protocol
described above. Quantities of the fluorescent tag-modified
peptides and the EGFR in each fraction are shown in Fig. 5.
The quantity of the EGFR was estimated from the fluores-
cence intensity at 333 nm (excitation at 280 nm). EGFR was
detected in fraction 7 (46.3 pmol) and fraction 8 (59.5 pmol) as
shown in Fig. 5(a). Free peptides were detected in the
subsequent fractions. Fluorescent tag-modified peptides were
also detected in fraction 7 (Fig. 5(b); magnified fractions 5–9 in
Fig. 5(a)). These results indicate that the peptides bind to
EGFR and that the EGFR-binding peptides can be
successfully detected by this method. Acd(S/T) (3.9 pmol),
Tmr(F/Y/W) (2.3 pmol), Mac(I/L/M) (1.2 pmol) and
Moc(G/A/V) (0.8 pmol) were detected in fraction 7. Other
peptides were less than 0.4 pmol. These results indicate that
specific peptides that bind to an EGFR can be successfully
differentiated by this method.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 761–763 | 763