Highly sensitive SERS quantification of the oncogenic protein c-Jun in cellular extracts

Article abstract of DOI:10.1021/ja405120x

A surface-enhanced Raman scattering (SERS)-based sensor was developed for the detection of the oncoprotein c-Jun at nanomolar levels. c-Jun is a member of the bZIP (basic zipper) family of dimeric transcriptional activators, and its overexpression has bee

Full text of DOI:10.1021/ja405120x

Communication
pubs.acs.org/JACS
Highly Sensitive SERS Quantification of the Oncogenic Protein c‑Jun
in Cellular Extracts
Luca Guerrini,†,‡ Elena Pazos,§ Cristina Penas,§ M. Eugenio Vazquez,§ Jose Luis Mascarenas,*,§
́
̃
and Ramon A. Alvarez-Puebla*,†,‡,⊥
†Departamento de Ingenieria Electronica, Universitat Rovira i Virgili, Avda. Països Catalans 26, 43007 Tarragona, Spain
‡Centro de Tecnologia Quimica de Cataluna, Carrer de Marcel·lí Domingo s/n, 43007 Tarragona, Spain
̃
§Departamento de Química Organ
́
ica and Centro Singular de Investigacion
C/Jenaro de la Fuente, s/n, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
⊥ICREA, Passeig Lluís Companys 23, 08010 Barcelona, Spain
́
en Química Biolox
́
ica e Materiais Moleculares (CIQUS),
S
* Supporting Information
limitations, including, among others, colorimetric sandwich
assays,14 protein binding microarrays,15 and chromatin
immunoprecipitation chips.16 All of these methods rely on
the use of relatively elaborated DNA constructs, and most of
them suffer from low sensitivity or selectivity. In this regard, we
have recently developed a sensing strategy to detect the DNA-
binding domain of c-Jun on the basis of the increase in
luminescence of a designed c-Fos receptor when forming the
coiled coil heterodimer with c-Jun.17 The method avoids the
use of DNA receptors, but its sensitivity is relatively low.
In recent years, surface-enhanced Raman scattering (SERS)
spectroscopy has emerged as one of the most powerful
analytical techniques for the fast and sensitive interrogation
of specific targets.18−20 SERS retains the rich chemical and
structural information provided by Raman spectroscopy but
overcomes its inherent limitation to the investigation of low
amounts of material by exploiting the enormous electro-
magnetic field enhancement resulting from the excitation of
localized surface plasmon resonances at nanostructured metallic
surfaces (mostly gold or silver).21,22 Additionally, the existence
of surface selection rules23 imposes different enhancements for
the Raman features depending on the symmetry of the
corresponding vibrational modes. This provides a valuable
tool to investigate possible preferential orientations of the
molecule onto the metal surface. However, while the direct
SERS measurement of the intrinsic Raman spectra of small
molecules is relatively straightforward, several limitations in
sensitivity, selectivity, and reproducibility often arise for
biological targets with high molecular weight, such as
proteins.24 A potential solution to detecting such large
biomolecules is based on the introduction into the sensing
system of Raman labels with intense and characteristic SERS
signals that undergo distinctive changes upon recognition of the
biomolecular target. Usually, Raman labels have been installed
at the metal substrate together with the biomolecule receptor,
and the analyte recognition event is revealed by intensity
changes of the reporter SERS signal.25−28 Alternatively, the
analyte recognition can induce characteristic spectral changes of
the label SERS profile, which usually arise from re-orientation
ABSTRACT: A surface-enhanced Raman scattering
(SERS)-based sensor was developed for the detection of
the oncoprotein c-Jun at nanomolar levels. c-Jun is a
member of the bZIP (basic zipper) family of dimeric
transcriptional activators, and its overexpression has been
associated with carcinogenic mechanisms in several human
cancers. For our sensing purpose, we exploited the ability
of c-Jun to heterodimerize with its native protein partner,
c-Fos, and therefore designed a c-Fos peptide receptor
chemically modified to incorporate a thiophenol (TP)
group at the N-terminal site. The TP functionality anchors
the c-Fos protein onto the metal substrate and works as an
effective SERS probe to sense the structural rearrange-
ments associated with the c-Fos/c-Jun heterodimerization.
ranscription factors are proteins that bind to specific
Tregulatory regions of the genes and thereby control the
initiation of their transcription by the RNA polymerase.1 It is
well known that many anomalous cell processes, including
abnormal cellular proliferations related to a neoplastic trans-
formation, are intimately related to the aberrant expression of
some of these transcription factors.2 This is the case with the
oncoprotein c-Jun, a component of the AP-1 transcription
complex that has been associated with carcinogenic mecha-
nisms in several human cancers.3−9 Indeed, it has been shown
that a large number of malignant transformations present
elevated levels of c-Jun expression,4,10,11 although not much
quantitative information has been reported to date (due to the
lack of appropriate analytical methods). c-Jun content varies
with the cellular line under study from nanomolar to
micromolar and, generally, overexpressions over 1 order of
magnitude during carcinogenic processes are commonly
reported.10 Therefore, a prompt and reliable detection of this
oncoprotein in its complex biological media may have great
diagnostic and therapeutic impact, and aid in further studies on
its biological role. Conventional methods for identification of
transcription factors, such as electrophoretic mobility shift
assays12 and DNase footprinting assays,13 are quite laborious
and time-consuming. Thus, many efforts are being devoted to
develop analytical tools capable of overcoming these
Received: May 26, 2013
Published: July 1, 2013
© 2013 American Chemical Society
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Journal of the American Chemical Society
Communication
of the label with respect to the plasmonic surface (i.e., surface
selection rules) and/or deformation of its molecular structure
(i.e., rearrangement of the electronic cloud).29 In this
Communication, we describe a SERS sensor for highly sensitive
detection of c-Jun on a bio-relevant aqueous media. The sensor
features the DNA binding domain of a natural c-Jun partner (c-
Fos) and a suitable SERS reporter attached at its N-terminus,
and allows the specific detection of minute amounts of the
target.
It is well known that in bZIP transcription factors like AP-1,
c-Jun and c-Fos dimerize via a leucine zipper domain, a process
which promotes the folding of the largely unstructured
monomeric species into α-helix secondary structures in the
homo-/heterodimers.30 We envisioned that this drastic
conformational change could be exploited for our sensing
purposes by designing a c-Fos peptide receptor chemically
modified by covalent attachment of a thiophenol (TP) group at
the N-terminal amino acid residue. The TP functionality not
only acts as a strong anchoring group for attaching the protein
onto the metal substrate via a covalent bond but it also provides
a very intense, highly reproducible, and fully vibrationally
characterized SERS spectrum that might effectively sense the
structural rearrangements associated to the c-Fos/c-Jun
heterodimerization. In this regard, the selection of a single
benzene ring as a direct molecular spring joining the c-Fos
protein with the metal surface, is expected to maximize the
possible mechanical deformations of the SERS probe structure
upon c-Jun complexation (i.e., maximum sensitivity). As c-Jun
receptor we chose a peptide featuring the dimerization (leucine
zipper) and basic region of the c-Fos DNA binding domain
(amino acids 138−193).17,31,32
Figure 1. (A) Schematic representation of the SERS substrate (AgNPs
over silanized glass slide) including the normalized extinction spectra
of AgNPs in solution and deposited on the glass substrate. A
representative SEM image of the film is also shown. (B) Outline of the
c-Fos/c-Jun dimerization on the metal surface and the resulting
deformation of the Raman label structure. c-Fos peptide sequence:
MKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSAL-
QTEIANLLKEKEKLW.
Silver nanoparticles (AgNPs), prepared via chemical
reduction with sodium citrate, were self-assembled on 3-
(aminopropyl)triethoxysilane-modified glass slides (Figure 1A)
and then functionalized with the TP-labeled c-Fos synthetic
derivative (c-FosTP). In a subsequent step, octanethiol, a
molecular probe characterized by its low Raman cross-
section,33 was assembled to fill in any gaps within the c-
FosTP monolayer. The alkanethiol surface functionalization is
intended, first, to minimize direct interactions of the large c-Fos
protein moiety with the metal and thereby induce the adoption
of a preferential upright position and, second, to prevent
nonspecific binding of the target c-Jun and the components of
the biological medium to the metal. The so-prepared sensing
platform was then stored in HEPES buffer before being
incubated with c-Jun (DNA binding domain, see Supporting
Information) solutions at different concentrations.
AgNPs film functionalized with c-FosTP shows the intense
SERS features characteristic of the TP molecule34 (Figure 2A)
such as the bands at 1585 cm−1, ascribed to the aromatic
ν(CC) modes, at 1075 cm−1, assigned to an in-plane ring
breathing mode coupled with ν(C−S), at 1022 and 998 cm−1,
both resulting from in-plane ring breathing vibrations, at 691
cm−1, attributed to C−H out-of-plane deformation, and at 417
cm−1, assigned to the ν(C−S) mode. Upon exposure of the c-
FosTP-metal substrate to c-Jun solutions in HEPES buffer (20
mM HEPES pH 7.6, 60 mM NaCl) at different concentrations,
a re-shaping of the SERS spectral profile takes place. The most
prominent changes are observed for the bands at 1075 and
1585 cm−1. This is not surprising since the vibrational modes
associated with these bands are known to show the largest
interfacial contribution to the change in polarizability of the
phenyl ring (Ph) in the TP−metal surface complex.35 Such
intrinsic property is at the heart of the largest enhancement and
marked spectral downshifts that these Raman features exhibit
upon adsorption of TP onto the metal.35,36 For the same
reason, the contributions at 1075 and 1585 cm−1 are truly
spectral markers for monitoring deformations of the electronic
molecular structure of the Ph−S−metal surface complex caused
by external factors such as binding events involving a
substituent group of the phenyl ring29 (as for the c-Fos/c-Jun
dimerization).
In the case of the in-plane ring breathing mode coupled with
ν(C−S), the corresponding Raman band exhibits a progressive
downshift with increasing c-Jun concentrations (Figure 2B),
whereas in the aromatic CC stretching region (Figure 2D) we
observe a drastic increase in the relative intensity of the weak
shoulder at ca. 1574 cm−1, corresponding to the non-totally
symmetric CC vibration,37 with respect to the totally symmetric
mode at 1585 cm−1.37 Such redistribution of the Raman
scattering cross sections among the aromatic ν(CC) vibrations
is theoretically attributed to the perturbation of the Cs
symmetry of the benzene ring of the Ph−S−Ag surface
complex.37 Additionally, the normalized SERS spectra illus-
trated in Figure 2A show a rather uniform intensity increase of
the in-plane vibrations at 417, 998, and 1022 cm−1 and of the
C−H out-of-plane deformation at 691 cm−1 with respect to the
two marker bands (Figure S2). The unspecific intensity
increase of these in-plane and out-of-plane modes suggests
that the overall spectral changes observed upon c-Jun addition
are largely determined by the modification of the electronic
polarizability of the Raman probe as a consequence of the c-
Fos/c-Jun dimerization, rather than by a mere re-orientation of
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Communication
correlated quantitatively with the c-Jun concentration using as
spectral markers (i) the peak position of the ring breathing and
ν(CS) feature (Figure 2C) and (ii) ratiometric peak intensities,
I1574(complex)/I1585(free) (Figure 2E), respectively. Spectral decon-
volution was performed for each Raman band between 1574
and 1585 cm−1 at fixed fwhm values with the assumption of
Lorentzian lineshapes.38 Higher sensitivity to low amounts of c-
Jun and better reproducibility from sample to sample are
observed in the plot of I1574(complex)/I1585(free) versus analyte
concentration, which shows a detection limit of 5 nM and r2 =
0.99 in the 1 × 10−9−5 × 10−6 M range, with a saturation point
at 5 × 10−6 M.
Importantly, c-Jun can also be clearly detected in cell extracts
(total protein concentration equals to 10 times in weight the c-
Jun concentration) albeit in this case the detection limit is
approximately 1 order of magnitude higher, but still very good
(DL = 50 nM, Figure 3). Careful optimization of the plasmonic
Figure 3. Intensity ratio I1574(complex)/I1585(free) at different c-Jun
concentrations in HEPES buffer and cell extracts. The X bar was
placed at Y = 0.55 which corresponds to the intensity ratio value
before the addition of c-Jun solutions. Error bars equal two standard
deviations (N = 3).
substrate, such as the reduction of the exposed c-FosTP-
modified metal surface area, or modification of the peptide
receptor with acidic sequences to increase affinity,17,39,40 should
allow to further improving the sensitivity of the system. We also
performed controls to assess the specificity of the SERS
measurements. Interestingly, addition of HEPES buffer
solutions of c-Fos (5 × 10−5 M), BSA (5 × 10−5 M), and
cell extracts (380 μg/mL) left unaltered the SERS spectrum of
c-FosTP, confirming the selectivity of the sensing strategy
(Figure S3).
Figure 2. (A) Normalized SERS spectra of c-FosTP anchored to
AgNPs over a silanized glass slide upon exposure to variable
concentrations of c-Jun in HEPES buffer. (B,D) Details of the
1000−1100 and 1540−1620 cm−1 spectral regions of the SERS spectra
illustrated in (A), respectively. (C) Spectral shift of the thiophenol
band at ca. 1075 cm−1 as a function of c-Jun concentration
(logarithmic scale) in HEPES buffer. (E) Intensity ratio I1574(complex)
/
In summary, we have engineered a highly sensitive SERS-
based sensor for the quantification of the oncoprotein c-Jun via
functionalization of a nanostructured silver substrate with a c-
Fos peptide receptor chemically modified by covalent attach-
ment of a thiophenol group. Selective heterodimerization of the
two proteins in the c-Fos/c-Jun complex triggers the molecular
deformation of the TP structure, as revealed by characteristic
changes in its SERS spectrum. Quantitative correlation between
spectral marker bands and oncoprotein concentration allowed
the detection of c-Jun down to the nanomolar regime in its
complex biological medium. Even though it is demonstrated for
the quantification c-Jun, potential applications of this sensing
strategy may include other macromolecular analytes, thus giving
rise to an extremely valuable tool in diagnosis and chemical
biology.
I1585(free) as a function of c-Jun concentration (logarithmic scale) in
HEPES buffer, with a limit of detection at 5 nM. Error bars equal to
two standard deviations (N = 3).
the benzene ring on the metal surface. The biomolecular
recognition event can therefore be schematically summarized as
depicted in Figure 1B; the protein c-FosTP exists in its
unstructured monomeric form in the self-assembled monolayer.
The presence of c-Jun promotes the formation of heterodimeric
coiled-coil complexes and therefore a drastic restructuring of
the geometry into a more rigid conformation. This structural
change leads to a sensible deformation of the phenyl ring
sandwiched in the c-Jun/c-Fos Ph−S−Ag surface complex,
which is immediately revealed by the spectral analysis of the
SERS data. The spectral changes illustrated in Figure 2B,D were
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Journal of the American Chemical Society
Communication
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ASSOCIATED CONTENT
■
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S
* Supporting Information
Experimental procedures, peptide synthesis and labeling
protocols, Raman spectra of proteins, analysis of SERS data,
SERS spectra of control experiments. This material is available
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AUTHOR INFORMATION
■
Corresponding Author
joseluis.mascarenas@usc.es; ramon.alvarez@urv.cat
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
́
This work was funded by the Spanish Ministerio de Economia
y Competitividad (CTQ2011-23167). We also acknowledge
the financial support provided by the Spanish grants SAF2010-
20822-C02, CTQ2012-31341, CSD2007-00006, Consolider
Ingenio 2010, the Xunta de Galicia GRC2010/12, INCITE09
209084PR, and PGIDIT08CSA-047209PR. C.P. thanks the
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