Rapid Screening of Peptide Probes through In Situ Single-Bead

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Rapid Screening of Peptide Probes through In Situ Single-Bead Sequencing Microarray Weizhi Wang,†,‡ Zewen Wei,†,‡ Di Zhang,†,‡ Huailei Ma,† Zihua Wang,† Xiangli Bu,† Menglin Li,† Lingling Geng,† Christopher Lausted,§ Leroy Hood,§ Qiaojun Fang,† Hao Wang,*,† and Zhiyuan Hu*,†,‡,§ †

CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, China ‡ Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China § Institute for Systems Biology, 401 Terry Avenue N., Seattle, Washington 98109, United States S Supporting Information *

ABSTRACT: Peptide ligands as targeting probes for in vivo imaging and drug delivery have attracted great interest in the biomedical community. However, high affinity and specificity screening of large peptide libraries remains a tedious process. Here, we report a continuous-flow microfluidic method for one-bead−one-compound (OBOC) combinatorial peptide library screening. We screened a library with 2 × 105 peptide beads within 4 h and discovered 140 noncanonical peptide hits targeting the tumor marker, aminopeptidase N (APN). Using the Clustal algorithm, we identified the conserved sequence Tyr-XX-Tyr in the N terminal. We demonstrated that the novel sequence YVEYHLC peptides have both nanomolar affinity and high specificity for APN in ex vivo and in vivo models. We envision that the successful demonstration of this integrated novel nanotechnology for peptide screening and identification open a new avenue for rapid discovery of new peptidebased reagents for disease diagnostics and therapeutics.

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number of beads can be sorted (see Scheme 1). A one-beadone-well microarray which is compatible with in situ matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) sequencing was also developed. This system has been applied to the discovery of noncanonical affinity peptide ligands from OBOC libraries toward the important tumor marker aminopeptidase N (APN). APN is a membrane protein that plays a key role in tumor angiogenesis.38 APN has been suggested as a valid target for anticancer therapy. Different approaches have been used to develop drugs for this target including enzyme inhibitors and an APN-targeted carrier.12,39 Hundreds of 7-mer affinity peptide ligands were identified effectively from the 2 × 105 OBOC candidates within 4 h. In vivo and ex vivo experiments have shown that these novel ligands have both high affinity and high specificity toward APN, which are prerequisites for the development of effective tumor probes.

olecular recognition by affinity ligands is increasingly employed in a wide range of biomedical fields such as in vivo imaging, disease diagnosis, and cancer therapy.1−4 Peptides are excellent small-molecule ligands, because of their convenient synthesis and their desirable biological properties such as good cell penetrability, low immunogenicity, and high biocompatibility.5−8 Various examples have been identified as tumor targeting ligands such as peptides with RGD9−11 or NGR12−14 for molecular imaging and drug delivery.15−17 Furthermore, peptide-based therapeutics is of extensive clinical use. 18−27 The combinatorial one-bead−one-compound (OBOC) library approach has been utilized to discover novel peptide ligands,28−30 yet conventional strategies to manually isolate bead hits from the millions of library beads are laborintensive and time-consuming. Therefore, an integrated system consisting of peptide selection, sorting, cleavage, and sequencing in a high-throughput manner is desired. Our previous efforts have focused on high-throughput peptide synthesis and screening using microfluidic chips.31−33 Based on the previous work of ours and others,34−37 we report here an integrated screening strategy based on a lab-on-chip system that embraces the entire peptide ligand screening process: highthroughput positive peptide isolation, sorting, single bead trapping, and in situ mass spectrometry (MS) sequencing and identification. Using a microfluidic chip with magnetic trapping and sheath flow sorting functions, a peptide library with a large © XXXX American Chemical Society

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EXPERIMENTAL SECTION Materials. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethy-

Received: September 15, 2014 Accepted: November 5, 2014

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synthesized through Fmoc strategy. The synthesized peptides were analyzed and purified by using a Hitachi HPLC system (L-7100, Japan) on a TSK gel ODS-100 V column (150 mm × 4.6 mm) at a flow rate of 1.0 mL min−1. Gradient: 0−25 min, 5−80% acetonitrile containing 0.1% TFA. The purified peptides were characterized by MALDI-TOF MS (Bruker Daltonics). In Situ MALDI-TOF Sequencing and Identification of the Peptide. MALDI-TOF MS analysis was performed on a Bruker ULTRAFLEXTREME mass spectrometer (Bruker Daltonics, Germany) equipped with a nitrogen laser (wavelength = 337 nm, laser pulse duration = 3 ns) with reflectron and positive-ion modes. The laser power energy was adjusted between 0% and 100% to provide laser pulse energy between 0 and 100 μJ per pulse. The mass spectra were typically recorded at an accelerating voltage of 19 kV, a reflection voltage of 20 kV, and with laser pulse energy of 60 μJ. Each mass spectrum was acquired as an average of 500 laser shots. For in situ “one well− one bead” analysis, the laser beam was directed to the bottom of the microwell with the light spot area of 0.003 mm2. Surface Plasmon Resonance Imaging (SPRi) Detection of the Four Affinity Peptides toward APN. SPRi analysis was performed on a Plexera PlexArray HT system (Plexera LLC, Bothell, WA) using bare gold SPRi chips (Nanocapture gold chips, with a gold layer of 47.5 nm thickness). All the purified peptides were adsorbed onto the gold chip surface and then incubated at 4 °C overnight in a humid box. The SPRi chip was washed and blocked using 5% (m/v) nonfat milk in PBS overnight before use. The SPRi analysis procedure operates using the following cycle of injections: running buffer (PBST, baseline stabilization); sample (five concentrations of the protein, binding); running buffer (PBST, washing); and 0.5% (v/v) H3PO4 in deionized water (regeneration). Protein (APN) was diluted with PBST to concentrations of 10, 5, 2.5, 1.25, and 0.625 μg/mL. Real-time binding signal were recorded and analyzed using a PlexArray HT system. SPRi is a label-free, real-time method to detect molecular interactions on metalcoated microarray surface. In Vitro Experiments of the Peptide Binding toward the Cells. Hepatoma cells HepG2 were cultured in a DMEM/ high glucose culture medium. Approximately 1 × 105 mL−1 cells were seeded into culture dishes and cultured overnight for the adhesion of cells. FITC-labeled peptide (AP-1, AP-2) was dissolved in cold PBS to a concentration of 1 mg/mL. After the cells had been washed with cold PBS, FITC-labeled peptide solution (200 μL) was added. Hoechst 33342 was spiked into the FITC-labeled peptide solution. Binding was achieved by incubating the cells in the solution for 60 min at 4 °C. Finally, the cells were washed three times with cold PBS. Confocal fluorescence imaging was performed on a confocal-laser scanning microscope (Olympus, Model FV1000-IX81). A Model FV5-LAMAR 488-nm laser was the excitation source for FITC throughout the experiment, and emission was collected between 520 nm and 620 nm. Hoechst 33342 was excited by a 50-mW, 405-nm Laser Head FV5-LD405-2 and collected with a band-pass filter within the range of 422−472 nm. The objective lens used for imaging were a UPLSAPO 100 × oil-immersion objective with a numerical aperture of 1.4 (Olympus). During the characterization of different samples, all of the parameters of the microscope were set to be the same for the comparison of the binding ability of the four peptides. In Vivo and Ex Vivo Fluorescence Imaging. All animal experiments were conducted in compliance with the guide for

Scheme 1. Principle of the Screening Process: (a) Overview of Chip System for Beads Trapping, Sorting, and HighThroughput In Situ Single Bead Sequencing; and (b) Positive Peptide Beads Will Be Surrounded by the Magnetic Beads through the Interaction Bridge of Peptide−APN− Biotin−Streptavidin, While Native Beads Will Remain Naked

luronium hexafluorophosphate (HBTU) were purchased from GL Biochem (China). Tentagel resin was obtained from Rapp Polymere (Germany). Trifluoroacetic acid (TFA), and fluorescein 5-isothiocyanate and streptavidin-coated magnetic beads (1 μm) were obtained from Sigma−Aldrich (USA). Silicon wafer (N/1−0−0, 500 μm) was from KYKY Tech. (China), APN protein was from Proteintech (USA), Mccoy’s 5A culture medium was obtained from Gibco and DMEM/ High glucose culture medium was obtained from Hyclone. Preparation of the OBOC Peptide Library and Resynthesis of the Affinity Peptides. Fmoc strategy SPPS (solid phase peptide synthesis) was employed for synthesis of the OBOC library.40 Tentagel resin (loading of 0.53 mmol/g) was used as the solid phase support. Scheme 2 shows the Scheme 2. Synthesis Process of the Photo Labile OBOC Peptide Library toward APN

synthesis process. During the OBOC library synthesis, solid support Tentagel beads were split equally in each cycle and different amino acids were added and then the beads were pooled together. (In the scheme, NMM represents N-methyl morpholine, DMF represents N,N-dimethylformamide, TFA represents trifluoroacetic acid, and SP represents the side chain protecting group.) The affinity leading peptide (AP-1) and another three peptides (AP-2, AP-3, and AP-4) were also B

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biotin−streptavidin conjugation, as shown in Scheme 1a. APN was biotinylated with an average of two biotins per molecule. The biotinylated APN was incubated with the OBOC library and then the 1 μm streptavidin-coated magnetic beads were loaded to search the biotinylated peptide beads. It was expected that the magnetic beads would associate with the peptide beads in an affinity-dependent manner. The ligands with the high affinity for the target protein could then be isolated using magnetic separation approaches in a continuous-flow microfluidic process (Scheme 1b). A photo cleavable linker, ANP (3amino-3-(2-nitrophenyl) propionic acid), was designed for in situ cleavage of the peptides from the beads. Microfluidic-Based Screening and Identification. Speed, throughput, and efficiency play key roles in OBOC peptide screening. We demonstrated here an automated and integrated microfluidic chip system that consisted of a beads sorting microchannel and in situ MALDI-TOF-MS sequencing microarray. The microfluidic chip system was fabricated using MEMS techniques on a silicon substrate. As shown in Scheme 1, after interaction, the mixture of the peptide library beads, biotinylated APN, and the magnetic beads were introduced into the microfluidic chip. A magnetic field was applied upstream in the channel. Target-binding, or positive, beads were coated by the magnetic beads and trapped while the negative beads would flow through. A sheath flow configuration was used to sort the positive beads and negative beads into different directions. In the downstream, each microwell had a cube shape of suitable size to trap individual peptide beads in a one-well−one-bead manner. The silicon chip was inserted into a modified MALDI target for in situ single-bead analysis. We obtained 140 sequences and aligned them as shown in Figure 1. Using the software ClustalX2,44 conserved sequences

the care and use of laboratory animals of Beijing University Animal Study Committee’s requirements. The Beijing University Animal Study Committee approved the experiments. The xenografted tumors were established by subcutaneously (s.c.) injecting 1 × 107 HepG2 cells into the right flank of the 4−5 week-old female BALB/c nude mice. Tumor size was measured periodically using calipers, and the tumors allowed to grow to 7−8 mm in diameter. Cy-5 labeled peptides (95% purity) were purchased from the Chinese Peptide Co., Ltd. Carboxyl group functionalized CdSe/ZnS core/shell quantum dots (QDs) with emission maxima centered at 687 nm were purchased from Invitrogen. The conjugation between the peptides and the QDs were carried out following the literature.41 Eight micromoles (8 μmol) of QDs sample (400 μL) in 1 × PBS was activated with 150 μmol of 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide (EDC, Sigma) and 7.5 μmol of N-hydroxysulfosuccinimide (sulfo-NHS, Sigma) for 2 h and then mixed with the 4-molar-excess peptides. There are 20−40 peptides per QD, based on a coupling efficiency of 40%−50%. Cy5-APs (1 μM, 200 μL) and QD-APs (1 μM, 200 μL) were injected into tumor-bearing nude mice via the tail vein and the tumor images were obtained using the small animal in vivo imaging system (CRI Maestro 2). Near-infrared fluorescence (NIRF) images of nude mice bearing subcutaneous tumor were acquired after vein injection of Cy5-AP-1, Cy5-AP-2, QD-AP-1, and QD-AP-2, while the control tumorbearing nude mouse was intravenously injected with Cy5 and QDs (1 μM, 200 μL). The mice were anesthetized and placed into the imaging system. NIRF images were taken to individually image the injectable Cy5 labeled peptides (ex: 488 nm ±10 nm, em: 694 nm ±17.5 nm, 10 s exposure) and the QDs labeled peptides (ex: 450 nm ±10 nm, em: 687 nm ±17.5 nm, 10 s exposure) signals, respectively. Then the nude mice were sacrificed and tumors as well as the main organs were harvested and also the NIRF images were taken individually.

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RESULTS AND DISCUSSION OBOC Peptide Library Design, Construction, and Interaction. The diversity of the structures and characters of the peptides are created by the different permutations of the amino acid sequence. In our strategy, mixes of designed or random amino acid residues were placed at specific positions to create a 7-mer OBOC combinatorial library for targeting APN. The peptide library was constructed with the sequence of X1X2X3X4X5X6C, in which X1 represents either F, Y, A, or L residues. Aromatic residues in the N-terminal have been reported to form a hydrophobic domain binding with APN.42,43 X2 and X3 represent V, E, I, or K residues as these hydrophilic and neutral residues may create good spatial structure for hydrophobic interaction in position X1. X4−6 represent N, R, L, D, G, S, H, or Y residues with different properties. As shown in Scheme 2, in the OBOC library, the peptide sequences on each bead were randomly distributed, so that the capacity of the peptide library was 8 × 8 × 8 × 4 × 4 × 4 = 32 768 and the redundancy of the library was six. Therefore, high-throughput peptide screening could be implemented with ∼2 × 105 candidate beads. The beads were monodispersed and had a diameter of 35 μm. Compared to conventional peptide screening, where sizes often exceed 200 μm, speed and throughput were greatly enhanced. In order to ensure an unbiased display for free molecular geometries, the interactions between the affinity peptide and APN protein is mediated by

Figure 1. Alignment of noncanonical APN-binding peptides using the ClustalX2 multiple alignment tool. Conserved motifs are highlighted.

were determined. It suggested that the motif Tyr-XX-Tyr, where X represents the hydrophilic amino acids residues, hit at the highest probability. The consensus sequence that emerged is YVEYHLC, referenced here as AP-1, with the most frequent matches among all seven residues. Another candidate ligand peptides YEKYHSC (AP-2) were chosen based on the frequency of matching residues. It is notable for this highthroughput screening system that promising sequences could be identified quickly by sequence alignment. Peptide Ligands as Probes for Imaging Cancer Cells In Vitro. To test the binding behavior of AP-1 and AP-2 toward APN protein in cancer cells, human cancer cell line, hepatoma cell HepG2 with high APN expression on the membrane was tested.45 To check the specificity of the selected peptides, a scrambled peptide (SP) with the sequence of VHLYYEC was served as a negative control. It was clear that AP-1 shows the C

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target on the complicated membrane structure. SKOV-3 cells with APN highly expression were employed as the model cells. APN siRNA was transfected into the cells and APN gene in SKOV-3 cells was suppressed by RNA interference mechanism (referred as RNAi SKOV-3). As expected, AP-1 preserves the binding ability toward SKOV-3 cells and fluorescent signal can be observed clearly in the membrane (see Figures 3a−d). For

highest signal (Figure 2a), followed AP-2 (Figure 2b) while SP shows little signal (Figure 2c). For quantitative comparison,

Figure 2. Fluorescence intensity and affinity comparison of the peptides: (a−c) Confocal image of FITC-labeled AP-1 (panel a), AP-2 (panel b), and SP (panel c) binding toward HepG2 cells. (d−f) The fluorescent profiles along the red arrow through the HepG2 cell membrane of AP-1 (panel d), AP-2 (panel e), and SP (panel f). (g−i) SPRi detection of the binding affinity of AP-1 (panel g), AP-2 (panel h), and SP (panel i) toward APN.

Figure 3. AP-1 peptide specifically targeted APN protein. (a−c) FITC-labeled AP-1 binds to SKOV-3 cells; (d) the fluorescent profiles along the red arrow through the SKOV-3 cell membrane; (e−g) FITC-labeled AP-1 binds to RNAi SKOV-3 cells; (h) the fluorescent profiles along the red arrow through the RNAi SKOV-3 cell membrane; (i−k) FITC-labeled AP-1 binds to 293A cells; (l) the fluorescent profiles along the red arrow through the 293A cell membrane; (m−o) FITC-labeled AP-1 binds to APN overexpression 293A cells; and (p) the fluorescent profiles along the red arrow through the APN overexpressed 293A cell membrane.

fluorescence intensity profiles along through the red arrow were also illustrated and AP-1 also showed highest signals (see Figures 2d−f). Dissociation constants between the peptide ligands and the APN protein were determined by SPRi. The dissociation constant was calculated from kinetic constants obtained by curve-fitting association and dissociation rates to real-time binding and washing data (see Figures 2g−i). The dissociation constants (KD) of the AP-1 and AP-2 were calculated to be 37.5 nmol/L and 452 nmol/L, respectively. These SPRi results agree nicely with the fluorescence results. Among them, the peptide ligand AP-1 illustrated a good binding affinity toward APN. The result is also in line with the high frequency appear in the in situ sequencing. To further confirm the theoretical binding affinity of AP-1 and AP-2 toward APN, interaction energy was calculated from computer simulation using MM/GBSA method.46 It shows that the predicted binding free energy of AP-1/APN, AP-2/APN, and SP/APN complex is −81.22 ± 4.09, −77.31 ± 3.94, and −67.91 ± 3.49 kcal/mol, respectively, which indicated that AP1 has the highest binding affinity toward APN. The simulation details were shown in the Supporting Information, and the binding energy is shown in Table S1 in the Supporting Information. In addition, decomposition of the total binding free energies into individual residue mode was done, to calculate each residue contribution (Figure S1 in the Supporting Information). The result shows that two Tyr in position 1 and position 4 of AP-1 and AP-2 in the N-terminal contributed the most in the binding toward APN. Confirmation of the Specificity of AP-1 for APN. These in vitro assays establish that the novel APs can recognize and bind to APN-expressing cancer cells. Further cell experiments have been carried out to confirm that whether APN is the exact

RNAi SKOV-3 cells, the decrease in green fluorescence can be discriminated easily, because of the low expression of APN protein (see Figures 3e−h). An APN overexpression cell model was also constructed in order to get further proof of the specific binding of the APs to APN. APN plasmid was transfected into the 293A cells and APN was specifically expressed on the membrane. FITC-labeled AP-1 was incubated with the common 293A cells and APN overexpression 293 cells. AP-1 demonstrated good binding ability toward APN overexpression 293 cells and fluorescent signal can be detected on the membrane (see Figures 3i−p). The binding mode of AP-2 and APN was similar to AP-1; the confocal image and quantification profiles are shown in Figure S2 in the Supporting Information. These results have further proved that the specific binding sites of the APs ligand are APN proteins. In Vivo and Ex Vivo Confirmation of the Tumor Homing Peptides. Advances within the field of in vivo optical imaging have added powerful tools for monitoring tumor development noninvasively in preclinical cancer models. The aforementioned results suggested that the cellular uptake of the probesparticularly, AP-1 and AP-2were facilitated through APN receptors, which encouraged us to further investigate the targeted tumor treatment in vivo. The biodistribution of peptide probes in tumor-bearing mice were monitored by NIR (nearD

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infrared) fluorescence imaging technique. Prior to in vivo studies, both of them were labeled with the Cy5 fluorophore or CdSe/ZnS core/shell QDs (quantum dots) independently, named Cy5-AP-1, Cy5-AP-2, QD-AP-1, and QD-AP-2. Two groups of separate subdermal tumor masses were identified through NIR imaging. Mice treated with intravenous injection of QD or Cy5 only were used as control. For the Cy5-labeled APs, as shown in Figures 4a and 4b, a half hour later, the

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CONCLUSION An efficient and fast strategy for screening peptide probes was developed. The affinity screening process is dramatically simplified and accelerated. As a model, our strategy was successfully applied to the identification of a peptide probe for aminopeptidase N (APN) protein not only in living cancer cells but also in vivo and ex vivo in mice model. The excellence of the AP1 peptide with high affinity and high specificity was demonstrated in in vitro, in vivo, and ex vivo models and makes it a promising targeting tool for cancer diagnosis and targetable drug delivery. In another experiment, good affinity peptides targeting HER2 were also discovered by this highthroughput system (data not shown). Our work provides a new insight into the establishment of effective and universal strategy for screening peptide probes for different biological system.

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ASSOCIATED CONTENT

S Supporting Information *

The free-energy calculation and the binding behavior of AP-2 toward cells are provided as Supporting Information. This material is available free of charge via the Internet at http:// pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-10-82545643. Fax: +86-10-82545643. E-mail: Zhiyuan Hu: [email protected]. *Hao Wang: [email protected]. Author Contributions ‡

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge funding from the National Natural Science Foundation of China (No. 21305023), Beijing Natural Science Foundation (No. 2144058), National Natural Science Foundation of China (Nos. 31270875, 31470049) and Project of Chinese Academy of Science (No. YZ201217). We express our gratitude to Dr. Yu Gao (Scripps Research Institute), Dr. Zongxiu Nie and Dr. Rui Zhao (Institute of Chemistry, Chinese Academy of Science), and Dr. Zhihong Li (Peking University) for their valuable help.

Figure 4. In vivo and ex vivo imaging of tumor targeting by AP-1 and AP-2: (a, c) in vivo fluorescence imaging of AP-1 to tumor (fluorescence was observed in both Cy5-labeled probes (panel a) and QD-labeled probes (panel c), whereas the targeted probes QDAP-1 presented with higher signal); (e, g) ex vivo fluorescence imaging of tumor accumulation and biodistribution; and (b, d, f, h) quantification of the fluorescence signals in vivo and ex vivo. Fluorescence intensity was measured in terms of counts/energy/area and is presented as an average (n = 3).

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fluorescence signal appeared at the tumor of which AP-1 display a higher signal than AP-2. This may be caused by the lower affinity of the AP-2 toward the APN. For the QD-labeled APs, AP-1 accumulated and retained preferentially in the tumor at 2 h post-injection (see Figures 4c and 4d). At 4 h, the animals were sacrificed, tumors were harvested and findings were validated ex vivo. As shown in Figures 4e and 4f, Cy5-AP-1 resulted in high levels of tumor accumulation and even abundant probes gathered in the kidney (overexposure in the figure). The ex vivo image also showed that fluorescence at the tumor site appears principally in QD-AP-1-injected nude mice. As shown in Figures 4g and 4h, consistent with results obtained using in vivo, the tumor showed the highest signal of 0.153 photons/cm2/s. These data demonstrated that the novel peptide probe AP-1 could correctly recognize the tumor and emit bright fluorescence in vivo, which confirmed the highest targeting specificity and excellent biocompatibility of all these probes.

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dx.doi.org/10.1021/ac503454z | Anal. Chem. XXXX, XXX, XXX−XXX