Photoaffinity casting of a coumarin flag for rapid identification of ligand-binding sites within protein

Article abstract of DOI:10.1039/c3cc38594a

A photo-switchable fluorescent flagging approach has been developed to identify photoaffinity-labeled peptides in target protein. Upon photochemical release of the ligand, the protein was newly modified with a coumarin in place of the previously attached biotin. It allowed us to simplify complex identification processes for labeled sites.

Full text of DOI:10.1039/c3cc38594a

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Photoaffinity casting of a coumarin flag for rapid
identification of ligand-binding sites within protein†
Shota Morimoto,a Takenori Tomohiro,*a Nobuyuki Maruyamab and
Yasumaru Hatanaka*a
Cite this: Chem. Commun., 2013,
49, 1811
Received 30th November 2012,
Accepted 11th January 2013
DOI: 10.1039/c3cc38594a
www.rsc.org/chemcomm
A photo-switchable fluorescent flagging approach has been developed
Several strategies have been investigated to facilitate the
to identify photoaffinity-labeled peptides in target protein. Upon identification of peptides, e.g., selective purification and detec-
photochemical release of the ligand, the protein was newly modified tion protocols. Purification has been achieved by introducing a
with a coumarin in place of the previously attached biotin. It allowed purification tag using a clickable group,4 a perfluoroalkyl chain,5
us to simplify complex identification processes for labeled sites.
and a biotin moiety.6 Detection has been performed by using
multifunctional cross-linkers bearing isotope-coded,7 fluorogenic,8
A high throughput photoaffinity-based method has been developed and cleavable functions.9 Thus far, diazirine-based biotinylated
for the identification of ligand-binding domains within proteins. probes have provided an efficient solution for the one-step purifi-
Structural elucidation as well as identification of the target protein cation of labeled proteins using an avidin-immobilized matrix,6
is a crucial matter in the field of life sciences for understanding and whereas peptide identification has been greatly affected by some
regulating complex biological processes. Photoaffinity cross-linking contaminants eluted from the matrix. Several scissile functional
with mass spectrometric analysis is a powerful approach to groups allow selective recovery of biotinylated products trapped
investigate the structure of proteins and their complexes.1,2 irreversibly on the matrix,10 while general cleavable approaches
This approach is particularly useful for analyzing proteins that inevitably result in the loss of biotin as a sensitive detection tag for
are difficult to analyze by conventional X-ray crystallography further assignment of the labeled peptide. Here, we report a
and solution-state NMR methods. The cross-linked proteins are unique and rapid method for identification of a protein domain
subjected to proteolytic digestion and the resulting peptide using a novel photo-cleavable biotin probe. This photoprobe tags a
fragments are analyzed by liquid chromatography-tandem fluorescent label in place of the previously attached biotin tag
mass spectrometry (LC-MS/MS). Despite the simplicity of the upon releasing a photoaffinity cross-linked protein.
cross-linking strategy, identification of cross-linked peptides is
To complete the identification of the labeled peptide by MS, the
often quite troublesome because of the complexity of the photoreactive unit was designed according to essential features
reaction mixtures and differences in the physical properties based on specificity of labeling, purification efficiency of labeled
of the peptides.1 3-Phenyl-3-trifluoromethyldiazirine has been proteins, selectivity and sensitivity for detection of labeled peptide
recognized as an ideal photophore that rapidly creates a stable fragments, and simplicity of handling. The cross-linker employed
covalent bond with any spatially closed molecule with minimal for this purpose was a newly developed o-hydroxycinnamate
diffusion.3 This is a crucial feature for site-specific labeling at diazirine compound.11 This molecule is a small-sized cross-linker
interfaces of physical interactions within proteins. However, in which only one double bond is added to a general phenyl-
regarding the identification of labeled peptide fragments, diazirine photophore, which prevents reduction in the affinity
a trace amount of inevitable adsorption could often greatly between the ligand and the receptor. This compound can perform
interfere with the purification at picomolar concentrations, and another photoreaction: E–Z photo-isomerization of the cinnamate
assignment of the labeled peptide by LC often fails.
group, which in turn induces lactonization via intermolecular
nucleophilic substitution by a hydroxy group at the ortho position
(Scheme 1).12 This reaction possesses 2 useful advantages. (1) It
releases the large ligand molecule from the cross-linked protein
under mild conditions, which enables the selective isolation of
protein fragments by using the biotin–avidin system. (2) It con-
currently forms a coumarin derivative at the ligand-binding site as
a stable fluorescent tag, which enables specific and highly sensitive
detection of the labeled products, without the further addition of a
a Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama,
2630 Sugitani, Toyama 930-0194, Japan. E-mail: ttomo@pha.u-toyama.ac.jp,
yasu@pha.u-toyama.ac.jp; Fax: +81 76-434-5063; Tel: +81 76-434-7516
b Laboratory of Food Quality Design and Development, Graduate School of
Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
† Electronic supplementary information (ESI) available: Materials and methods,
inhibition assay, fluorescence spectra, and additional MS and NMR data. See
DOI: 10.1039/c3cc38594a
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1811--1813 1811

View Article Online
Communication
ChemComm
Fig. 1 Structure of photoreactive VSD photoprobe 1 and chemiluminescence
detection of the GmVSR cross-linked with photoprobe 1. Photoreaction was
performed under 365 nm light at 0 1C for 0, 1, 5, 10, 30 min (panel A) in the
absence (lanes 1–6) or in the presence of an inhibitor (SSILRAFY) in various
equivalent mol ratios (lanes 7–9, panel B, samples were irradiated for 10 min).
Scheme 1 Tag-switching strategy for the identification of target proteins by
double photoreactions of a multifunctional cross-linker.
detection tag. Our photophore labeling system also eliminates the
necessity of labeling post cleavage and purification. Moreover,
compared to a large adduct, a minimally bulky coumarin tag
proves to be advantageous for higher resolution mass spectro-
scopic analysis of the labeled peptide. Briefly, the multifunctional
photoprobe can specifically capture and isolate the target protein
only by UV-irradiation and easily enable identification of the
labeled peptide fragment.
In this study, we applied this concept to the identification of
the substrate-binding domain of the vacuolar sorting receptor
(VSR) of soybean. During seed maturation, VSR, a membrane
protein, transports proteins such as newly synthesized seed
Fig. 2 GmVSR detected by the chemiluminescence method (A) for biotinylated
products, fluorescence method (B) for coumarin-labeled products, and CBB
staining. Emission from the band was detected through a band filter (420 nm,
fwhm 10 nm) under UVA irradiation.
storage proteins from the endoplasmic reticulum to the protein inhibitory patterns compared to previous results obtained from
storage vacuole via a transport vesicle.13 Recently, a C-terminal sorting experiments of green fluorescence protein fused with
peptide of soybean (Glycine max) 7S globulin (b-conglycinin) b-conglycinin C-terminal peptides (see the ESI†).15 These results
was identified as a vacuolar sorting determinant (VSD).14,15 indicate that specific labeling of photoprobe 1 at the VSD-
Further, we found that the C-terminal sequence (SSILRAFY) of binding domain was achieved. The yield of cross-linking was
the b-conglycinin a0 subunit is a binding partner for soybean about 5%, which was determined by comparison with the
VSR (Kd = 100–200 nM). The photoreactive unit was conjugated emission intensity of biotinylated BSA.
to the N-terminus of the peptide via biocytin to affix the photo-
The second photoreaction was subsequently carried out with
probe 1 (Fig. 1, see the ESI† for Experimental details). Photo- 315 nm light (15 W) at 25 1C. The emission intensity of the
affinity labeling of a recombinant luminal soybean VSR (GmVSR, ligand-cross-linked GmVSR band decreased with the irradiation
1 mM) by photoprobe 1 (1 mM) was carried out with 360 nm light time in response to the release of a biotin group (Fig. 2A). At the
(a 30 W black light lamp) in a buffered solution containing same time, the intensity of fluorescence due to the coumarin
20 mM Hepes (pH 7.0), 150 mM NaCl, 1 mM CaCl2, 0.4% chaps, fluorophore increased at the same position (Fig. 2B). The result
and 0.02% NaN3 at 0 1C. Photoproducts were then subjected to of Coomassie Brilliant Blue (CBB) staining indicated that photo-
10% SDS–PAGE. The labeled products were detected by chemi- degradation of GmVSR did not occur during the irradiation
luminescence methods using horseradish peroxidase (HRP)- (Fig. 2C). The intramolecular nucleophilic substitution by a
conjugated avidin after blotting onto a PVDF membrane because hydroxy group proceeded efficiently at 25 1C to create a coumarin
the products were biotinylated. The emission intensity of the derivative. While some of the cross-linked GmVSR allowed
band around 55 kDa increased depending on the irradiation coumarin formation during the denaturing process at room
time (Fig. 1A) and clearly decreased in the presence of SSILRAFY, temperature (lane 1, Fig. 2B), the 2 reactions of cross-linking
which served as an inhibitor (Fig. 1B). In addition, competitive and cleavage were well regulated by changing irradiation wave-
binding assays were performed with other VSD analogs, in which length and reaction temperature.
1 or 2 amino acid residues were substituted with glycine. These
We then applied this fluorogenic photo-cleavage reaction
peptides had reduced affinity to GmVSR, which exhibited similar for purification of the labeled protein. After removal of the
c
1812 Chem. Commun., 2013, 49, 1811--1813
This journal is The Royal Society of Chemistry 2013

View Article Online
ChemComm
Communication
display bn + 240 u, yn + 240 u series, containing a coumarin
modification (Fig. 3B). There are y3 + 240 u, y4 + 240 u ions, and
unmodified y2 ions, as well as b3 + 240 u ions. The spectrum
demonstrated that the coumarin modification occurred on the valine
residue at the 3rd position from the N-terminus of the fragment.
In conclusion, the photochemical coumarin tagging method
allowed us to identify the labeled peptides rapidly. The requi-
site amount of protein for analysis is in the 10 micrograms
range. In addition, this approach can identify multiple peptide
components of a binding domain that could not be charac-
terized by conventional photolabeling approaches. The label-
switching strategy coupled with high-selective purification
should give a great advantage for identification of the target
proteins of low abundance, which is an inherent problem in
techniques such as shotgun proteomics.16
This research was supported by Grants-in-Aid for Scientific
Research (20390032, 21310138) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
Notes and references
1 (a) B. Domon and R. Aebersold, Science, 2006, 312, 212; (b) A. Sinz,
Mass Spectrom. Rev., 2006, 25, 663; (c) K. J. Pacholarz, R. A. Garlish,
R. J. Taylor and P. E. Barran, Chem. Soc. Rev., 2012, 41, 4335;
(d) T. Lenz, J. J. Fischer and M. Dreger, J. Proteomics, 2011, 75, 100.
2 M. A. Trakselis, S. C. Alley and F. T. Ishmael, Bioconjugate Chem.,
2005, 16, 741.
3 (a) J. Brunner, Annu. Rev. Biochem., 1993, 62, 483; (b) A. Blencowe
and W. Hayes, Soft Matter, 2005, 1, 178; (c) J. Das, Chem. Rev., 2011,
111, 4405; (d) J. Wang, J. Kubicki, H. Peng and M. S. Platz, J. Am.
Chem. Soc., 2008, 130, 6604.
4 (a) C. X. Song and C. He, Acc. Chem. Res., 2011, 44, 709; (b) C. H.
Sohn, H. D. Agnew, J. E. Lee, M. J. Sweredoski, R. L. J. Graham, G. T.
Smith, S. Hess, G. Czerwieniec, J. A. Loo, J. R. Heath, R. J. Deshaies
and J. L. Beauchamp, Anal. Chem., 2012, 84, 2662; (c) A. L.
MacKinnon and J. Taunton, Curr. Protoc. Chem. Biol., 2009, 1, 55;
(d) M. Hashimoto and Y. Hatanaka, Chem. Pharm. Bull., 2005, 53, 1510.
5 Z. Song, W. Huang and Q. Zhang, Chem. Commun., 2012, 48, 3339.
6 (a) T. Tomohiro, M. Hashimoto and Y. Hatanaka, Chem. Rec., 2005,
5, 385; (b) Y. Hatanaka, M. Hashimoto and Y. Kanaoka, Bioorg. Med.
Chem., 1994, 2, 1367; (c) Y. Luo, C. Blex, O. Baessler, M. Glinski,
Fig. 3 (A) Reverse-phase HPLC profiles of the digested products showing lysyl
endopeptidase detected by absorption at 215 nm (upper) and fluorescence at
420 nm (lex = 320 nm). (B) Tandem MS/MS spectrum of the [FVVEK + H]+ ion
(peak 1, m/z 861.50 in Fig. S3B, ESI†).
non-cross-linked probe by dialysis, the cross-linked products
were subjected to an avidin-immobilized agarose gel. After
washing with 0.2% SDS-containing PBS solution several times,
the gel was exposed to 315 nm light for one hour at 25 1C. Pure,
labeled GmVSR was recovered from the solid matrix in 60%
yield, which was estimated using CBB staining. The labeled
product showed fluorescent emission due to the corresponding
coumarin. This reaction was mild, selective, and efficient, and
yielded almost pure protein without contamination with other
adsorbed materials.
After digestion of the labeled GmVSR with lysyl endopeptidase,
the resulting peptides were separated by reverse-phase HPLC. As
shown in the HPLC profiles (Fig. 3A), and compared to the result
detected by UV absorption at 215 nm, there were a limited
number of peaks, which were detected easily with high sensitivity
using emission at 420 nm (lex = 320 nm). These peaks appeared
identical to the labeled peptides because of the presence of
¨
M. Dreger, M. Sefkow and H. Koster, Mol. Cell. Proteomics, 2009,
8, 2843.
7 (a) K. K. Palaniappan, A. A. Pitcher, B. P. Smart, D. R. Spiciarich,
A. T. Iavarone and C. R. Bertozzi, ACS Chem. Biol., 2011, 6, 829;
(b) M. Hashimoto and Y. Hatanaka, Chem. Pharm. Bull., 2004,
52, 1385.
8 (a) E. W. Chan, S. Chattopadhaya, R. C. Panicker, X. Huang and
S. Q. Yao, J. Am. Chem. Soc., 2004, 126, 14435; (b) T. Kuroda,
K. Suenaga, A. Sakakura, T. Handa K. Okamoto and H. Kigoshi,
Bioconjugate Chem., 2006, 17, 524.
9 (a) Y. Chen, Y. W. Ebright and R. H. Ebright, Science, 1994, 265, 90;
(b) J. J. Park, Y. Sadakane, K. Masuda, T. Tomohiro, T. Nakano and
Y. Hatanaka, ChemBioChem, 2005, 6, 814; (c) A. F. Gomes and F. C. J.
Gozzo, J. Mass Spectrom., 2010, 45, 892.
10 G. Leriche, L. Chisholm and A. Wagner, Bioorg. Med. Chem., 2012,
20, 571, and references therein.
coumarin. This is one of the most beneficial things in this method 11 T. Tomohiro, K. Kato, S. Masuda, H. Kishi and Y. Hatanaka,
Bioconjugate Chem., 2011, 22, 315.
12 A. D. Turner, S. V. Pizzo, G. Rozakis and N. A. Porter, J. Am. Chem.
from a standpoint of skipping the hampered assignment pro-
cesses. Two major peaks indicated by arrows were identified by
Soc., 1988, 110, 244.
ESI-MS as the coumarin-modified FVVEK (Dm/z = 240.0 u) 13 (a) T. Shimada, K. Fuji, K. Tamura, M. Kondo, M. Nishimura and
(861.4 for MH+ + coumarin, found 861.5) and VWNAQK
I. Hara-Nishimura, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 16095;
(b) A. Vitale and N. V. Raikhel, Trends Plant Sci., 1999, 10, 316.
14 K. Nishizawa, N. Maruyama and S. Utsumi, Plant Mol. Biol., 2006,
(985.4 for MH+ + coumarin, found 985.0). The results of MS
identification from labeling were reproducible. These peptides
are derived from the N-terminal region at the 2nd to 6th residues
and 85th to 90th residues, respectively. The tandem mass
spectrum acquired for the single-charged [FVVEK + H]+ ion
62, 111.
15 K. Nishizawa, N. Maruyama, R. Satoh, Y. Fuchikami, T. Higasa and
S. Utsumi, Plant J., 2003, 34, 647.
16 P. Picotti, R. Aebersold and B. Domon, Mol. Cell. Proteomics, 2007,
6, 1589.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 1811--1813 1813