Peptide Microarray with Ligands at High Density Based on

May 16, 2014 - Microarray system including SmartArrayer 48 microarray spotter, BioMixer II microarray hybridization station, Slide Washer, and LuxScan...
0 downloads 0 Views 396KB Size
Download PDF
Article pubs.acs.org/ac

Peptide Microarray with Ligands at High Density Based on Symmetrical Carrier Landscape Phage for Detection of Cellulase Huan Qi,†,‡ Fei Wang,†,‡ Valery A. Petrenko,§ and Aihua Liu*,†,‡ †

Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China ‡ University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China § Department of Pathobiology, Auburn University, 269 Greene Hall, Auburn, Alabama 36849-5519, United States S Supporting Information *

ABSTRACT: Peptide microarrays evolved recently as a routine analytical implementation in various research areas due to their unique characteristics. However, the immobilization of peptides with high density in each spot during the fabricating process remains a problem, which will affect the performance of the resultant microarray greatly. To respond to this challenge, a novel peptide immobilization method using symmetrical phage carrier was developed in this work. The cellulytic enzyme endoglucanase I (EG I) was used as a model for selection of its specific peptide ligands from the f8/8 landscape library. Three phage monoclones were selected and identified by the specificity array, of which one phage monoclone displaying the fusion peptide EGSDPRMV (phage EGSDPRMV) could bind EG I specifically with highest affinity. Subsequently, the phage EGSDPRMV was used directly to construct peptide microarray. For comparison, major coat protein pVIII fused EG I specific peptide EGSDPRMV (pVIII-fused EGSDPRMV) which was isolated from phage EGSDPRMV was also immobilized by traditional method to fabricate peptide microarray. The fluorescent signal of the phage EGSDPRMV-mediated peptide microarray was more reproducible and about four times higher than the value for pVIII-fused EGSDPRMV-based microarray, suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. Further, the phage EGSDPRMV based microarray not only simplified the procedure of microarray construction but also exhibited significantly enhanced sensitivity due to the symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides in each spot. Thus, the proposed strategy has the advantages that the immobilizing peptide ligands were not disturbed by their composition and the immobilized peptides were highly regular with free amino-terminal.

T

he protein microarray, which emerged in the 1990s,1 has attracted much attention due to its high throughput, affordability, sensitivity, and specificity. Peptide microarray which evolved recently as an advanced variant of the protein array has played an important role in many fields,2 such as epitope mapping, 3 identifying substrates of interesting proteins,4 optimizing the composition of substrates,5 measuring the activities of enzymes,6 screening the ligands7 and inhibitors,8 evaluating the binding mechanisms of peptides and other molecules,9 and detecting pathogen infections.10 Many researchers over the world have demonstrated that the density of ligand in each spot could affect its biological properties.11,12 For most peptide microarrays, however, each peptide molecule has to be physically linked with an active group on the surface of slide, which may lead to distortion of the peptide active binding conformation and decrease the density of the peptide in each spot. In addition, the immobilization of peptides on the slides can be affected by their composition, especially when lysine13 or cysteine residues9 are located in the middle of the peptide sequence or binding © 2014 American Chemical Society

sites. Recently, some new models for increasing the density of ligands in each spot were developed, such as site-specific immobilization,14−20 three-dimensional slides, and dendrimers.21 To date, although some improvements in peptide/ matrix coupling have been achieved, some extra amino acids had to be introduced or peptides to be modified, which was often laborious and nonefficient. Herein, we proposed a potent peptide immobilization method mediated by symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides. The Ff phage (f1, fd, or M13) is composed of a singlestranded circular DNA and five coat proteins to form cylindral structure (Scheme 1). As shown in Scheme 1A, coat proteins pIII and pVI (each five copies) are located in the terminal of Ff phage, while coat proteins pVII and pIX (each five copies) are located in the other tip.22 pVIII, the major capsid protein, in Received: February 13, 2014 Accepted: May 16, 2014 Published: May 16, 2014 5844

dx.doi.org/10.1021/ac501265y | Anal. Chem. 2014, 86, 5844−5850

Analytical Chemistry

Article

(New Jersey, U.S.). Other chemicals were supplied by the standard suppliers. Instrumentation. The SDS-PAGE was run on the Biorad Mini-PROTEAN Tetra Electrophoresis System (Hercules, California, U.S.), and corresponding image was acquired on the ImageScanner III from GE Healthcare (Piscataway, New Jersey, U.S.). Microarray system including SmartArrayer 48 microarray spotter, BioMixer II microarray hybridization station, Slide Washer, and LuxScan 10K-A microarray scanner was purchased from CapitalBio Corporation (Beijing, China). The software LuxScan 3.0 provided by CapitalBio Corporation (Beijing, China) was used to extract and interpret data from slides. Selection of EG I Binding Phage Clones. The general procedures for amplification, purification, and titration of phage, including the phage monoclones and sublibraries, were reported by one of our co-workers.38 The preparation of starved cells was carried out by using the method Brigati JR et al. described with minor modification.39 The procedure of selection was carried out as described by one of our co-workers with modification.40 As shown in the Scheme S1 (Supporting Information), 50 μL of target solution (10 μg/mL) in tris-buffered saline (TBS) was put into selected wells of a 96-well high-binding plate from NUNC (Roskilde, Denmark). After being centrifuged at 4 °C (550 g, 2 min), the plate was transferred to a cold room with gently rocking for 16 h. Microwell containing the target was emptied and blocked with 100 μL of 10 g/L bovine serum albumin (BSA) for 1 h at room temperature. After blocking solution was aspirated, the microwell was washed 10 times with 100 μL of TBS containing 0.5% Tween 20 for 10 min every time to remove any unbound target and BSA. The f8/8 phage library (about 1011 virions in 50 μL of TBS containing 0.1% BSA and 0.5% Tween 20) was put into the above exhausted microwell and incubated in a cold room for another 16 h with gentle shaking. Nonbound phage particles were removed by washing 10 times with 100 μL of TBS containing 0.5% Tween 20 for 10 min every time. The bound phage particles were eluted by incubating with 100 μL of elution buffer (0.1 mol/L HCl containing 1 mg/mL BSA and 0.1 mg/mL phenol red, for which pH was adjusted to 2.2 with glycine) for 10 min at room temperature with gentle rocking. The eluate was aspirated to a microcentrifuge tube, neutralized with 19 μL of 1 M Tris-HCl (pH 9.1), and concentrated to about 50 μL with a Centricon 30-kD unit. These phage particles were output phages in first round selection. A few of these concentrated phage particles were used for determining the titer and monitoring the procedures of selections, while the remaining phages were amplified and purified for next round of biopanning and indicated in the sublibrary. In second round selection, the sublibrary, not the primary phage library, was used to further be screened. The remaining procedures were the same as in the first round. After 3 rounds, 10 phage clones were picked up with sterile toothpick for sequencing and amplifying. According to the method developed by one of the coauthors,38 the fragments of phage genome containing the information on displayed octapeptide were first amplified by PCR, whose products were detected by 1% agarose gel electrophoresis and sequenced by the BGI Inc. (Shenzhen, China). Finally, the displayed octapeptide sequences were deduced from corresponding sequence results. Phage Capture Assay. Phage capture assay was executed as described by one of our co-workers with modification.40 As

Scheme 1. (A) Structure of Phage fd. (B), f8/8 Landscape Phage (Not to Scale)

several thousand copies, surrounds the ssDNA to form a fivefold helical symmetry shield (Scheme 1A) protecting DNA from environmental stresses.22 The unique structural characteristics make the Ff phage very stable in many extreme conditions, such as extreme pH (pH 2.2−12, 37 °C, 30 min), presence of urea (60 °C, 60 min) or guanidine hydrochloride (37 °C, 90 min),23,24 and presence of nonwater media.25 Due to the symmetrical structure of Ff phage, the fusion foreign peptides are exposed symmetrically (Scheme 1B) on the surface of the phage if the type VIII display system is adopted to construct the library. As a typical representative of the type VIII display system, the f8/8 landscape library26 was used to select ligands with high affinity and high specificity for many different targets27−29 and found wide sensing applications.30 In addition, besides the extreme conditions mentioned above, the landscape phage could stand high temperatures.31 Therefore, in this project f8/8 landscape library was used as the source for isolating phage particles specific to cellulase and loading them onto the microarray matrixes. The use of a phage carrier that allows to load preselected genetically fused peptides symmetrically in extremely organized order, which is advantageous in comparison with the use of commercially available phage display peptide libraries, such as the Ph.D.-12 or Ph.D.-7 phage, display peptide library from the NEW ENGLAND BioLabs Inc. or other peptides screening systems.32,33 These libraries can be helpful in identification of binding peptides but in contrast to landscape phage cannot recruit phage particles that can be directly used as nanomaterials for generation of interfaces in different detection devices.34,35 Given the importance of cellulase in bioconversion from lignocellulytic biomass to biofuels,36 the endoglucanase I (EG I) isolated from the Trichoderma reesei QM9414 was chosen as a model to obtain specific ligands from the f8/8 landscape library.26 The performance of peptide microarray mediated by peptides displayed by landscape phage carrier was compared with coupled pVIII fused EG I specific peptides. Lastly, the more detailed characteristics of phage peptide microarrays were investigated.

■

EXPERIMENTAL SECTION Materials and Reagents. The f8/8 landscape library was developed by one of our coauthors. The Nexterion H with Nhydroxysuccinimide (NHS)-activated slides were purchased from Schott (Mainz, Germany). Endoglucanase I (EG I) and other five cellulolytic enzymes had been purified in our lab.37 The Cy3 Ab labeling kit was purchased from GE Healthcare 5845

dx.doi.org/10.1021/ac501265y | Anal. Chem. 2014, 86, 5844−5850

Analytical Chemistry

Article

shown in Scheme S2 (Supporting Information), 50 μL of each enzyme in TBS (10 μg/mL) was applied to different wells of a plate. The plate was centrifuged, incubated, blocked, and washed as above-mentioned. Isolated phages (about 106 TU in 50 μL of TBS containing 0.1% BSA and 0.5% Tween 20) were added to each well containing different enzymes. The following steps were the same with the above selection protocol, and the titers of eluates from each well were determined directly. Proteins Labeled with Fluorescent Dye. The EG I from Trichoderma reesei QM9414 and BSA were labeled with Cy3 according to the instruction of the labeling kit from GE healthcare. The excess free dye was removed by gel filtration. Optimization of the Titer of Phage EGSDPRMV. After purification, the phage EGSDPRMV was dissolved in 50 mM phosphate buffer (pH 7.4) and stored at 4 °C before use. The concentration of phage EGSDPRMV for constructing microarray was optimized. As shown in Scheme 2, different titers of

isolated from phage EGSDPRMV. Briefly, the solution of phage EGSDPRMV was mixed thoroughly with equal volume of saturated phenol (pH 8.0) for 8 min. After being centrifuged at 3000 g for 10 min, the upper aqueous solution was discarded. The remains were re-extracted four more times with equal volume of 1 M Tris-HCl (pH 8.0). The obtained peptide-fused major coat protein in the phenol was mixed with two volumes of methanol. The mixture was then serially dialyzed against the following solutions: the solution of methanol mixed with equal volume of 10 mM Tris-HCl (pH 8.0), the solution of methanol mixed with three times volumes of 10 mM Tris-HCl (pH 8.0) and deionized water (dialysis against each solution lasted for 12 h). Finally, the pVIII-fused EGSDPRMV was lyophilized, identified by electrophoresis,43 and stored in −20 °C for subsequent experiments. Peptide Microarrays Based on Both Landscape Phage EGSDPRMV and pVIII-Fused EGSDPRMV. To examine the efficiency of microarray mediated by landscape phage EGSDPRMV, two peptide microarrays both containing the peptide sequence EGSDPRMV were constructed, of which one array was mediated by the symmetrical carrier landscape phage EGSDPRMV at the optimized titer as in the above-mentioned method and another array was produced through immobilizing the pVIII-fused EGSDPRMV. The lyophilized pVIII-fused EGSDPRMV was redispersed in ion-free water at 2 mg/mL and then mixed thoroughly with equal volume of the above spotting buffer. The following procedures of fabricating, blocking, washing, incubating, and scanning were the same as the above method. Data Analysis. All data were extracted by LuxScan 3.0 and processed by the method described by Oda Stoevesan et al.44 The error bars were calculated as the standard deviations of the repeated spots in the same microarray.

Scheme 2. Construction of Phage-Based Peptide Microarray (Not to Scale)

■

RESULTS AND DISCUSSION

Selection of EG I Binding Phage Clones from f8/8 Landscape Library. Phages specifically binding enzyme EG I were selected from f8/8 landscape library. During the selection procedures, the recovery rate of binding phages was increased gradually at each consecutive round in the selection procedure (Supporting Information, Figure S1), which indicated that more and more phage clones existing in the sublibraries were capable of binding to EG I. After three rounds of biopanning, 10 clones were picked up, and their fragments of DNA containing segment of gene gpVIII were amplified by PCR (Supporting Information, Figure S2), purified, sequenced, and translated into the sequence of displayed peptides. Three unique amino acid sequences (Supporting Information, Table S1) of peptide ligands were revealed in 10 phage monoclones. Phage Capture Assay. Specificity and affinity of selected phages toward different cellulytic enzymes were studied by phage capture assay. As shown in Figure 1A, all three phages interacted with enzyme EG I. Compared with phage DRSVNTQT and phage DRVATSAPA in family 1, however, the affinity of phage EGSDPRMV toward enzyme EG I was stronger. In addition, the component in family 2 only interacted with enzyme EG I, while both phages in family 1 interacted with other enzymes. Therefore, the peptide EGSDPRMV with good affinity and specificity to EG I was chosen as the candidate ligand in construction of peptide microarrays. Furthermore, the phage selected from f8/8 landscape library, allowing to display the specific peptide EGSDPRMV sym-

phage solution were mixed with equal volumes of spotting buffer (300 mM phosphate buffer with 0.005% Tween 20, pH 8.5) and then spotted on the NHS-activated slides by microarray spotter at room temperature. Each sample was repeated several times with a distance of 300 μm between the centers of adjacent spots. At the same time, BSA-Cy3 (BSA labeled with Cy3), 50 mM phosphate buffer (pH 7.4), and phage fd-tet were also spotted on individual slides as positive, blank, and negative control, respectively. When the printing was completed, the printed slides were put in a 75% humidity chamber at 37 °C for immobilization. After completion, the slides were stored in vacuum until required or used immediately. The slides were immersed in the blocking buffer to quench the residual active group and then washed three times with phosphate buffered saline (PBS, pH 7.4) containing 0.1% Tween 20 for 5 min each time.41 After being washed with ionfree water once more, the microarray was probed with EG ICy3 (EG I labeled with Cy3) at 37 °C for 2 h in a microarray hybridization station and washed as in the above-mentioned method. Finally, the slides were dried by centrifugation and scanned at 532 nm using a LuxScan 10K-A microarray scanner with 800 photomultiplier gain and 100% of the laser power. Isolation of pVIII-Fused EGSDPRMV from Phage EGSDPRMV. According to the reported procedures42 with some modifications, the pVIII-fused EG I-specific peptide EGSDPRMV (abbreviated as pVIII-fused EGSDPRMV) was 5846

dx.doi.org/10.1021/ac501265y | Anal. Chem. 2014, 86, 5844−5850

Analytical Chemistry

Article

Figure 2. Optimization of phage probe concentration. Up graph, image of the array after incubation with 100 nM EG I-Cy3. P, positive spots; B, blank; N, negative spots; all following rows, different titers of phage probe. Bottom graph, the quantitative analysis of results of the left image.

Figure 1. Affinity and specificity of selected phages DRSVNTQT (A), DRVATSPA (B), and EGSDPRMV (C), respectively (here and after phages are designated by the sequences of inserted foreign peptides). CBH I, II and EG I, II were the abbreviation of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, and endoglucanase II, respectively. No values in the phage recovery indicated no colony existed in the plate. In other words, the number of phage in corresponding elution was about less than 40 TU.

metrically in a dense pattern on the phage surface, was also obtained for comparison. Optimization of the Titer of Candidate Phage EGSDPRMV. The peptide EGSDPRMV was immobilized on the surface of the slide mediated by the symmetrical carrier, landscape phage. As shown in Scheme 2, the amino groups of N-terminal alanine preceding the inserted peptide EGSDPRMV would react readily with NHS-functionalized slide.3 According to the procedures of the f8/8 landscape library,26 the size of phage EGSDPRMV should be similar to the phage fd-tet, which is about 1300 nm in length and 6.5 nm in width (Supporting Information, Figure S3). Since there are up to 4000 copies of peptide displayed symmetrically on each monoclonal landscape

Figure 3. Tricine-SDS-PAGE electrophoresis results of purified protein, of which lane M was protein marker and lanes 1, 2, and 3 were loaded 1, 0.5, and 0.25 μg of purified protein, respectively.

phage,45 it is not necessary that each peptide have a physical linkage with an active group of each spot on the surface of the 5847

dx.doi.org/10.1021/ac501265y | Anal. Chem. 2014, 86, 5844−5850

Analytical Chemistry

Article

Figure 4. (A) Pattern of fabricated microarray, in which 1 indicated the pVIII-fused EGSDPRMV and 2 indicated phage EGSDPRMV. (B) Image of peptide microarray incubated with 100 nM EG I-Cy3. (C) Quantitative results of image B. Figure 5. Fluorescence intensity−dose curve of the peptide microarray mediated by phage EGSDPRMV. (B) Image of peptide microarray mediated by phage EGSDPRMV after incubation with 100 nM EG ICy3.

slide during immobilization. In other words, once landscape phage was immobilized, a lot of peptides on the phage surface were also immobilized, which would dramatically increase the density of peptides in each spot. In addition, due to the fivefold rotational symmetrical distribution of the peptide ligands on the surface of Ff phage (Scheme 1B), a majority of displayed peptides are always far away from the surface of the slide and amino groups in the recombinant pVIII protein cannot react with the functional groups on the slide. Therefore, these displayed peptides would arrange away from the slide surface and retain their native conformation and free amino terminal groups. Other groups had also developed the peptide chips using peptides discovered Ff phage,46−48 T7 phage,49 and herpes virus displayed system;50 nevertheless, according to the properties of these display systems, the copy numbers of displayed proteins or peptides were small, which led to the low density immobilization. To investigate the feasibility of this approach and optimize the concentration of phage EGSDPRMV in conjugation reaction, according to the procedures shown in Scheme 2, the peptide microarray mediated by symmetrical carrier landscape phage was constructed by the method described in the Experimental Section. As shown in the seven rows from the bottom (Figure 2, up graph), the spots containing phage EGSDPRMV could be visualized by staining with 100 nM EG I-Cy3, which indicated that phage displayed peptides after phage immobilization retained their native conformation and ability to bind target molecules, which proved the efficiency of the phage-based strategy for peptide immobilization. Control experiments (the spots located in the third row on up graph of

Figure 2) demonstrated that the peptide EGSDPRMV played a very important role in binding enzyme EG I. As demonstrated in the bottom graph of Figure 2, the fluorescence intensity was increased in a dose-dependent manner with the concentration of phage. However, it was difficult to prepare a phage solution with concentration higher than 3 × 1012 transforming unit (TU)/mL by culture due to phage aggregation. In addition, the background noise can also be increased with the increasing concentration of phage probe. Therefore, the concentration of phage 2.2 × 1012 TU/mL was used as optima in subsequent experiments. Comparison of the Efficiency of Microarray Mediated by Phage EGSDPRMV and pVIII-Fused EGSDPRMV. Plenty of our previous results had demonstrated that the peptide fusion pVIII isolated from the landscape phage could retain its perfect respective specific target-binding functions,27 such as binding streptavidin51 and interacting with MCF-7 cells52 and PC3 cells.53 Therefore, to compare the performances of the peptide microarray mediated by landscape phage and peptide directly, the pVIII-fused EGSDPRMV was isolated from the phage EGSDPRMV. The tricine-SDS-PAGE electrophoresis of purified protein was performed, and a band with the molecular weight of about 8 kDa was obtained (Figure 3), which was nearly the theoretical molecular weight (5.8 kDa) of pVIII-fused EGSDPRMV. The difference in molecular weight is probably originating from the conformation of pVIII-fused 5848

dx.doi.org/10.1021/ac501265y | Anal. Chem. 2014, 86, 5844−5850

Analytical Chemistry

Article

relationship is shown in Figure 5A. The fluorescence intensity was linear with EG I concentration within 5−500 nM EG I (R 2 = 0.9960). Further, all spots from the phage-based peptide microarray were highly uniform in morphology, size, and signal intensity (Figure 5B), demonstrating high reproducibility of the phage microarray. In contrast, the spots of the peptide microarray were usually varied in morphology, size, and signal intensity, which resulted from the random direction and inactivation of probes during immobilization.54

EGSDPRMV, because the pVIII-fused EGSDPRMV is a membrane-associated protein, which was not boiled with loading buffer in the tricine-SDS-PAGE to avoid any aggregation. The amino acid sequence of the pVIII-fused EGSDPRMV is NH2-AEGSDPRMVDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKAS-COOH, of which the underlined portion is the displayed peptide of phage EGSDPRMV. Subsequently, arrays immobilized with both the phage EGSDPRMV and pVIII-fused EGSDPRMV were fabricated as described above (Scheme 2) and incubated with 100 nM EG ICy3. The spots were uneven and fluorescent signal was weak for pVIII-fused EGSDPRMV-based array (Figure 4B, left array). In contrast, spots with much stronger signal were observed for phage EGSDPRMV-mediated peptide array (Figure 4B, right array). Actually, the fluorescent signal for phage EGSDPRMV-mediated peptide array was about four times higher than the value for pVIII-fused EGSDPRMV-based array (Figure 4C), suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. These contrasting results can be explained in different ways. First, the density of peptide with free alanine in each spot is an important factor. As shown in Scheme 2, an active NHSgroup on the slide can theoretically immobilize many peptides with intact configuration in the microarray mediated by phage EGSDPRMV. However, only two or more NHS groups were necessary to immobilize pVIII-fused EGSDPRMV with uncertain configuration. In addition, the structural characteristics of the phage-displayed peptides, which were presented in high density and native conformation at the surface of phage, were still in high density after the corresponding phage was immobilized on the surface of slide. Further, the majority of immobilized peptide was native and functional. Therefore, the avidity effect of these peptides might be another important factor. Finally, the pVIII-fused EGSDPRMV can be immobilized to the slide in different forms: through either alanine or lysine, or both, the two amino acids to generate nonhomogeneous binding interface in each spot, while most of peptides fused to the phage retained their original targetbinding configuration. A quantitative model could also be used to further demonstrate the high efficiency of peptide immobilization based symmetrical carrier. The optimal concentration of phage EGSDPRMV in immobilization conditions was 2.2 × 1012 TU/ mL, corresponding to the 146 μM peptide EGSDPRMV (Supporting Information, Formula S1), most of which might be immobilized with the free amino-terminal and maintained in high density. In contrast, the concentration of pVIII-fused EGSDPRMV was 2 mg/mL, or about 172 μM, which was higher than the concentration of phage-displayed peptide. That is, some of the immobilized peptides by pVIII-fused EGSDPRMV might become inactive due to nonhomogeneous binding through either alanine or lysine or both of the two amino acids, which would be make it difficult to keep the high density of specific ligand with free amino-terminal, even if the concentration of peptide was increased further. Therefore, this quantitative model further suggested the high efficiency of peptide immobilization mediated by symmetrical phage carrier. Analytical Performance of the Peptide Microarray Mediated by Phage EGSDPRMV. The response of peptide microarray fabricated at the optimized concentration of phage EGSDPRMV was studied as a function of enzyme EG I concentration. The fluorescence intensity−enzyme dose

■

CONCLUSIONS In summary, we developed a novel peptide immobilization method using landscape phage as a symmetrical peptide carrier, which made the immobilized peptides at high density in each spot and regular direction and avoided the disturbances from their composition. In addition, the use of f8/8 landscape libraries for selection of the specific peptide ligand binding to protein analyte of interest and obtaining phage-displayed peptide symmetrically for being applied in the slide interface can be executed simultaneously, which significantly simplified the procedure of microarray construction and production. Thus, given the high flexibility and wide application of the phage display platform, this peptide immobilization strategy can be of unsurpassed utility in the field of peptide microarrays.

■

ASSOCIATED CONTENT

S Supporting Information *

Schemes of experimental procedures of selection of ligands from f8/8 landscape library, phage capture test, results about biopanning, electrophoresis of PCR products, TEM image of fd-tet phage, and formula to calculate peptide concentration. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interests.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (91227116, 31200598, and 21275152), the Hundred-Talent-Project (KSCX2-YW-BR-7), and 135 Project Fund of CAS-QIBEBT Director Innovation Foundation, Chinese Academy of Sciences.

■

REFERENCES

(1) Fodor, S. P.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767−773. (2) Min, D. H.; Mrksich, M. Curr. Opin. Chem. Biol. 2004, 8, 554− 558. (3) Martinez-Botas, J.; Cerecedo, I.; Zamora, J.; Vlaicu, C.; Dieguez, M. C.; Gomez-Coronado, D.; de Dios, V.; Terrados, S.; de la Hoz, B. Int. Arch. Allergy Immunol. 2013, 161, 11−20. (4) Sun, H. Y.; Lu, C. H. S.; Uttamchandani, M.; Xia, Y.; Liou, Y. C.; Yao, S. Q. Angew. Chem., Int. Ed. Engl. 2008, 47, 1698−1702. (5) Hilpert, K.; Hansen, G.; Wessner, H.; Schneider-Mergener, J.; Hohne, W. J. Biochem. 2000, 128, 1051−1057. (6) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270−274. (7) Wang, Z. X.; Laursen, R. A. Peptide Res. 1992, 5, 275−280. 5849

dx.doi.org/10.1021/ac501265y | Anal. Chem. 2014, 86, 5844−5850

Analytical Chemistry

Article

(8) Dostmann, W. R.; Taylor, M. S.; Nickl, C. K.; Brayden, J. E.; Frank, R.; Tegge, W. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14772− 14777. (9) Pai, J.; Yoon, T.; Kim, N. D.; Lee, I. S.; Yu, J.; Shin, I. J. Am. Chem. Soc. 2012, 134, 19287−19296. (10) Gaseitsiwe, S.; Valentini, D.; Mahdavifar, S.; Magalhaes, I.; Hoft, D. F.; Zerweck, J.; Schutkowski, M.; Andersson, J.; Reilly, M.; Maeurer, M. J. PLoS One 2008, 3, e3840. (11) Houseman, B. T.; Mrksich, M. Angew. Chem., Int. Ed. Engl. 1999, 38, 782−785. (12) Kato, M.; Mrksich, M. Biochemistry 2004, 43, 2699−2707. (13) Tessier, P. M.; Lindquist, S. Nature 2007, 447, 556−562. (14) Winssinger, N.; Harris, J. L.; Backes, B. J.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 2001, 40, 3152−3155. (15) Sijbrandij, T.; Cukkemane, N.; Nazmi, K.; Veerman, E. C.; Bikker, F. J. Bioconjugate Chem. 2013, 24, 828−831. (16) Lesaicherre, M. L.; Uttamchandani, M.; Chen, G. Y. J.; Yao, S. Q. Bioorg. Med. Chem. Lett. 2002, 12, 2079−2083. (17) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 11790−11791. (18) Wu, H.; Ge, J.; Yang, P. Y.; Wang, J.; Uttamchandani, M.; Yao, S. Q. J. Am. Chem. Soc. 2011, 133, 1946−1954. (19) Wammes, A. E. M.; Fischer, M. J. E.; de Mol, N. J.; van Eldijk, M. B.; Rutjes, F. P. J. T.; van Hest, J. C. M.; van Delft, F. L. Lab Chip 2013, 13, 1863−1867. (20) Tao, S. C.; Zhu, H. Nat. Biotechnol. 2006, 24, 1253−1254. (21) Benters, R.; Niemeyer, C. M.; Wohrle, D. ChemBioChem. 2001, 2, 686−694. (22) Chung, W. J.; Oh, J. W.; Kwak, K.; Lee, B. Y.; Meyer, J.; Wang, E.; Hexemer, A.; Lee, S. W. Nature 2011, 478, 364−368. (23) Kristensen, P.; Winter, G. Folding Des. 1998, 3, 321−328. (24) Tuna, M.; Woolfson, D. N. Engineering protein folding and stability. In Phage Display in Biotechnology and Drug Discovery; Sidhu, S. S., Ed.; CRC Press/Taylor & Francis: Boca Raton, FL, 2005; pp 385− 414. (25) Olofsson, L.; Ankarloo, J.; Andersson, P. O.; Nicholls, I. A. Chem. Biol. 2001, 8, 661−671. (26) Petrenko, V. A.; Smith, G. P.; Gong, X.; Quinn, T. Protein Eng. 1996, 9, 797−801. (27) Petrenko, V. A.; Jayanna, P. K. FEBS Lett. 2014, 588, 341−349. (28) Lang, Q.; Wang, F.; Yin, L.; Liu, M.; Petrenko, V. A.; Liu, A. Anal. Chem. 2014, 86, 2767−2774. (29) Jayanna, P. K.; Bedi, D.; Deinnocentes, P.; Bird, R. C.; Petrenko, V. A. Protein Eng., Des. Sel. 2010, 23, 423−430. (30) Mao, C. B.; Liu, A. H.; Cao, B. R. Angew. Chem., Int. Ed. Engl. 2009, 48, 6790−6810. (31) Brigati, J. R.; Petrenko, V. A. Anal. Bioanal. Chem. 2005, 382, 1346−1350. (32) Esvelt, K. M.; Carlson, J. C.; Liu, D. R. Nature 2011, 472, 499− U550. (33) Ishizawa, T.; Kawakami, T.; Reid, P. C.; Murakami, H. J. Am. Chem. Soc. 2013, 135, 5433−5440. (34) Petrenko, V.; Smith, G. P.; O’Brien, P.; Craighead, H.; Kroto, H. Phage Nanobiotechnology; Royal Society of Chemistry: London, U.K., 2011. (35) Qi, H.; Lu, H.; Qiu, H.-J.; Petrenko, V.; Liu, A. H. J. Mol. Biol. 2012, 417, 129−143. (36) Jarvis, M. Nature 2003, 426, 611−612. (37) Qi, H.; Bai, F.; Liu, A. Biochemistry (Moscow) 2013, 78, 424− 430. (38) Sorokulova, I. B.; Olsen, E. V.; Chen, I. H.; Fiebor, B.; Barbaree, J. M.; Vodyanoy, V. J.; Chin, B. A.; Petrenko, V. A. J. Microbiol. Methods 2005, 63, 55−72. (39) Brigati, J. R.; Samoylova, T. I.; Jayanna, P. K.; Petrenko, V. A. Curr. Protoc. Protein Sci. 2008, 51, 18.9.1−18.9.27. (40) Brigati, J.; Williams, D. D.; Sorokulova, I. B.; Nanduri, V.; Chen, I. H.; Turnbough, C. L.; Petrenko, V. A. Clin. Chem. 2004, 50, 1899− 1906.

(41) Blixt, O.; Head, S.; Mondala, T.; Scanlan, C.; Huflejt, M. E.; Alvarez, R.; Bryan, M. C.; Fazio, F.; Calarese, D.; Stevens, J.; Razi, N.; Stevens, D. J.; Skehel, J. J.; van Die, I.; Burton, D. R.; Wilson, I. A.; Cummings, R.; Bovin, N.; Wong, C. H.; Paulson, J. C. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17033−17038. (42) Knippers, R.; Hoffmanb, H. J. Mol. Biol. 1966, 21, 281−292. (43) Schagger, H. Nat. Protoc. 2006, 1, 16−22. (44) Stoevesandt, O.; Kohler, K.; Wolf, S.; Andre, T.; Hummel, W.; Brock, R. Mol. Cell. Proteomics 2007, 6, 503−513. (45) Samoylova, T. I.; Petrenko, V. A.; Morrison, N. E.; Globa, L. P.; Baker, H. J.; Cox, N. R. Mol. Cancer Ther. 2003, 2, 1129−1137. (46) Khattar, N. H.; Coe-Atkinson, S. P.; Stromberg, A. J.; Jett, J. R.; Hirschowitz, E. A. Cancer Biol. Ther. 2010, 10, 267−272. (47) Yoo, S. Y.; Oh, J. W.; Lee, S. W. Langmuir 2012, 28, 2166− 2172. (48) Bi, Q.; Cen, X. D.; Wang, W. J.; Zhao, X. S.; Wang, X.; Shen, T.; Zhu, S. G. Biosens. Bioelectron. 2007, 22, 3278−3282. (49) Babel, I.; Barderas, R.; Diaz-Uriarte, R.; Moreno, V.; Suarez, A.; Fernandez-Acenero, M. J.; Salazar, R.; Capella, G.; Casal, J. I. Mol. Cell. Proteomics 2011, 10, M110.001784. (50) Hu, S.; Feng, Y.; Henson, B.; Wang, B.; Huang, X.; Li, M.; Desai, P.; Zhu, H. Anal. Chem. 2013, 85, 8046−8054. (51) Jayanna, P. K.; Torchilin, V. P.; Petrenko, V. A. Nanomed.: Nanotechnol., Biol. Med. 2009, 5, 83−89. (52) Bedi, D.; Gillespie, J. W.; Petrenko, V. A.; Ebner, A.; Leitner, M.; Hinterdorfer, P.; Petrenko, V. A. Mol. Pharmaceutics 2013, 10, 551− 559. (53) Bedi, D.; Musacchio, T.; Fagbohun, O. A.; Gillespie, J. W.; Deinnocentes, P.; Bird, R. C.; Bookbinder, L.; Torchilin, V. P.; Petrenko, V. A. . Nanomed.: Nanotechnol., Biol. Med. 2011, 7, 315−323. (54) Samanta, D.; Sarkar, A. Chem. Soc. Rev. 2011, 40, 2567−2592.

5850

dx.doi.org/10.1021/ac501265y | Anal. Chem. 2014, 86, 5844−5850