Design and Optimization of a Phosphopeptide Anchor for Specific

Nov 3, 2014 - The attachment of affinity proteins onto zirconium phosphonate coated glass slides was investigated by fusing a short phosphorylated pep...
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Design and Optimization of a Phosphopeptide Anchor for Specific Immobilization of a Capture Protein on Zirconium Phosphonate Modified Supports Hao Liu,† Clémence Queffélec,*,‡ Cathy Charlier,§ Alain Defontaine,§ Amina Fateh,§ Charles Tellier,§ Daniel R. Talham,*,† and Bruno Bujoli‡ †

Department of Chemistry, University of Florida, Gainesville, Florida 32611-7200, United States Chimie Et Interdisciplinarité: Synthèse Analyse Modélisation (CEISAM), Université de Nantes, CNRS, UMR 6230, 2, rue de la Houssinière, BP 92208, 44322 Nantes, Cedex 3, France § Fonctionnalité et Ingénierie des Protéines (UFIP), Université de Nantes, CNRS, UMR 6286, 2, rue de la Houssinière BP 92208, 44322 Nantes, Cedex 3, France ‡

S Supporting Information *

ABSTRACT: The attachment of affinity proteins onto zirconium phosphonate coated glass slides was investigated by fusing a short phosphorylated peptide sequence at one extremity to enable selective bonding to the active surface via the formation of zirconium phosphate coordinate covalent bonds. In a model study, the binding of short peptides containing zero to four phosphorylated serine units and a biotin end-group was assessed by surface plasmon resonance-enhanced ellipsometry (SPREE) as well as in a microarray format using fluorescence detection of AlexaFluor 647-labeled streptavidin. Significant binding to the zirconated surface was only observed in the case of the phosphopeptides, with the best performance, as judged by streptavidin capture, observed for peptides with three or four phosphorylation sites and when spotted at pH 3. When fusing similar phosphopeptide tags to the affinity protein, the presence of four phosphate groups in the tag allows efficient immobilization of the proteins and efficient capture of their target.

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INTRODUCTION DNA microarrays are now widely used for the high throughput analysis of biological media,1,2 but there remain major challenges to finding universal methods for applying biological arrays to other fields, such as proteomics and medical diagnosis, among others.3−7 Following the success of oligonucleotide microarrays,8,9 there is no doubt that successful platforms will enjoy tremendous application and become routine in the near future, but a key point in the development of such biosensors is control and optimization of the binding of the biological probes on solid supports to obtain integrated analytical and miniaturized devices.10−12 The binding of the probes needs to be efficient and with homogeneous orientation to facilitate the capture of analytes and increase the sensitivity of detection.13−15 To make devices of practical use, strong binding between the probe and the support is desirable, using chemical processes that are as simple and inexpensive as possible but robust enough to ensure consistency in applications. A variety of methods are commonly used for immobilizing proteins for microarray studies,16,17 most often based on nonspecific adsorption of the protein to a solid support, for example via H-bonding or electrostatic interactions.18 Simple © 2014 American Chemical Society

chemical coupling of reactive groups present on proteins, typically amine or carboxylic acid residues, have also been used to immobilize proteins onto surfaces suitably modified by complementary functional groups.19,20 Both methods, which require highly purified proteins, often result in randomly oriented and partially denatured proteins. More recently, the use of recombinant tags allows proteins to attach to substrates in a defined orientation, but these more specific interactions, based, for example, on glutathione S-transferase or oligohistidine, are often reversible.21,22 In this context, we are interested in developing general methods for immobilizing proteins and other biomolecules requiring little or no chemical modification of the probes and using easily accessible microarray substrates. A few years ago, we reported the use of zirconium phosphonate-coated glass slides as reactive surfaces able to provide covalent attachment of phosphate-terminated biological probes.23−27 This approach is very attractive since phosphorylation can be achieved using enzymatic methods without affecting the functionality of the Received: September 11, 2014 Revised: November 3, 2014 Published: November 3, 2014 13949

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methane refractive index matching fluid, and α-casein were used as received from Aldrich. All other reagents were of analytical grade and were used as received from commercial sources, unless otherwise indicated. Glass slides were purchased from Goldseal (Horseshoe, NC) (3 in. × 1 in., thickness 0.93−1.05 mm). Aminopropylsilane glass slides (SuperAmine 2) were purchased from Arrayit (Sunnyvale, CA). Slides used for SPREE experiments were made of 28.5 nm of gold evaporated on a 4 nm chromium adhesion layer on a clean SF10 glass slide (Schott Glass). Biotinylated phosphorylated peptides were purchased from ProteoGenix (Schiltighem, France). The buffer used throughout the experiments consisted of 10 mM glycine, 10 mM TRIS base, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 10 mM acetic acid, allowing buffering in the range pH = 2−10. Zirconium Phosphonate-Modified Slides. Zirconium phosphonate-coated glass slides were prepared by adapting a published procedure.28 SuperAmine 2 slides (used as received) were soaked in a 1:1 solution of 2,4,6-collidine (20 mM) and POCl3 (20 mM) in anhydrous acetonitrile and gently rocked for 16 h. The slides were rinsed with acetonitrile and then annealed at 200 °C for 24 h to promote the formation of a stable phosphonate coating. The phosphonate-terminated samples were then exposed to a 25 mM ZrOCl2·8H2O solution in Milli-Q water overnight. The slides were rinsed with Milli-Q water and stored under argon in Milli-Q water. Production and Purification of Tagged Proteins. H4 Nanofitins are recombinant proteins derived from Sac7d from Sulfolobus acidocaldarius and evolved by ribosome display to bind lysozyme. The sequence of H4 Nanofitin was fused with the phosphorylatable tag (ARAEREDSDSSSEDE) containing four sites of phosphorylation in the plasmid pQE30H4P.21 Recombinant pQE30H4P1ala plasmid containing H4 phosphorylatable tag (ARAEREDADAASEDE) with one serine were derived by site directed mutagenesis from pQE30H4P. For protein production, BL21(DE3) E. coli strain transformed with these plasmids were cultivated in 1 L of Luria-Bertani (LB) medium containing ampicillin (100 μg/mL) at 37 °C until the optical density at 600 nm was 0.8. Isopropyl-1-β-D-thio-1-galactoside (IPTG) (1 mM) was added for induction and cells were grown overnight at 30 °C. The proteins were first purified on Ni-NTA columns (QIAGEN) and then phosphorylated in vitro with recombinant α-casein kinase (CKII). The Nanofitins (100 μM) were incubated for 6 h at 30 °C in 50 mM TrisHCl, 50 mM MgCl2, 1 μg of alpha CK2, 1 mM ATP, 2.5 mM DTT, 0.01% Triton X100, 10% glycerol, and 125 mM imidazole at pH 7.5, and the purification was completed by gel filtration on Superdex 75 (GE Healthcare). Microarray Spotting and Incubation Conditions. The modified slides were washed once with ultrapure water and dried by centrifugation (1500 rpm, 1 min). The slides were then spotted with a noncontact spotter (sciFlexArrayer S3 piezo electric dispenser, Scienion). Spotting was performed inside a chamber at 25 °C and 60% humidity, and then the spotted slides were placed in an incubation chamber at 37 °C for 1 h. To passivate the unspotted areas, slides were treated after spotting with a solution of 0.3 wt % α-casein in a TBS (Tris-buffered saline) solution of TrisHCl (20 mM) and NaCl (150 mM) at pH 7.4. The probes were exposed to fluorescent targets for analysis. In the case of spotted biotinylated phosphopeptides, incubation was performed by applying an Alexa 647-labeled streptavidin solution (0.1 μg/mL in TBS−0.3% α-casein) to the substrate for 1 h at room temperature in the dark. In the cases of the mono- or tetraphosphorylated tags fused to proteins, incubation was performed by applying an Alexa 647-labeled lyzozyme solution (0.1 μg/mL in TBS−0.3% α-casein) to the substrate for 1 h at room temperature in the dark. The microarrays were washed 3 times with TBS for 5 min and then once with ultrapure water. Finally, the slides were spun-dry by centrifugation at 1500 rpm for 1 min. All washes and incubations were performed in small staining jars at room temperature on an oscillating shaker. Microarray Analysis. All microarrays were scanned on a Scanarray Gx apparatus (PerkinElmer) with a laser power and gain value of 60. Suitable excitation wavelength and emission filter were used to detect Alexa 647: 650 nm (excitation) and 665 nm (emission). The location of each analyte spot on the array and measurement of the fluorescence

modified biological probes. This original concept was first demonstrated in the case of 5′-phosphate-terminated oligonucleotides for which specific terminal phosphate binding to the zirconium phosphonate surface was observed.27 The idea was then successfully extended to the immobilization of phosphate terminated ds-DNA.26 More recently, our goal has been to investigate the potential of this technology for the design of protein microarrays. It becomes more challenging, however, to ensure stable surface attachment of proteins while controlling their orientation. We recently introduced, in an exploratory study, the idea of attaching protein probes onto a zirconium phosphonate surface via a short phosphorylated peptide sequence fused at the N- or C-terminus of the proteins that are capable of forming “zirconium−(OPO3-peptide)” covalent bonds (Figure 1).21

Figure 1. Covalent attachment of proteins on zirconium phosphonate surfaces via phosphopeptide anchors.

In the present work, the best conditions for achieving high binding affinity of short phosphopeptides onto the zirconium phosphonate support are investigated, in particular by varying the pH and the number and location of phosphorylated serine units in the peptide sequence. In all cases the phosphopeptides were found to specifically bind on the zirconium phosphonate surface. However, when applied to affinity proteins bearing a terminal phosphorylated peptide tag, the presence of four phosphate binding groups was found to yield better sensitivity and signal-to-noise ratio in microarray studies.

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MATERIALS AND METHODS

Materials. Zirconyl chloride octahydrate, 2,4,6-collidine, phosphorus oxytrichloride (POCl3), octadecyltrichlorosilane, the diiodo13950

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intensities was performed using the Genepix mapping software (Axon laboratories, Palo Alto, CA). Surface Plasmon Resonance-Enhanced Ellipsometry (SPREE). SPREE measurements were performed on a commercial EP3-SW imaging system (Nanofilm Surface Analysis, Germany). The ellipsometer employed a frequency-doubled Nd:YAG laser (adjustable power up to 20 mW) at 532 nm. Linearly p-polarized light was directed through a 60° equilateral SF10 prism coupled to a gold-coated SF10 slide via diiodomethane index matching oil in the Kretschmann configuration.29 The angle of incidence was kept between 64° and 66° for all kinetics experiments because this condition provided the highest sensitivity. Curve fitting of the kinetics data used the AnalysR software (Nanofilm), using a 1:1 Langmuir binding model. SPR slides were prepared by rendering the gold-coated slides hydrophobic with octadecylmercaptan before transferring the zirconium phosphonate layer. The slides were placed in the SPR flow cell, and peptides binding onto the zirconium phosphonate surface were carried out by injecting 100 μL of 1 mg/mL peptide solutions in the buffer stream at the corresponding pH. The surface was regenerated using the same buffer as the peptide solvent.

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RESULTS 1. Phosphopeptide Binding on Zirconium Phosphonate Surfaces. 1.1. Spotting Experiments. Zirconium phosphonate layers on glass slides were prepared by adapting a published approach.28 Briefly, commercially available aminecoated slides (SuperAmine 2) were treated with POCl3 and subsequently dipped into an aqueous solution of zirconium oxychloride. The resulting zirconium phosphonate surfaces were then used to investigate the surface binding of short phosphopeptide segments, 12 amino acids, relative to a nonphosphorylated analogue in array spotting experiments. The number of phosphate groups on the peptide sequence was varied from zero to four, determined by the number of phosphorylatable serine moieties in the sequence (Table 1),

Figure 2. Fluorescence analysis of zirconium phosphonate substrates spotted with peptide sequences containing zero (0P), one (1P), two (2P), three (3P), or four (4P) phosphorylated serine groups (see Table 1), at different pHs. For each condition, three spotting concentrations were used (from left to the right: 10, 5, and 1 μM) in triplicate. After spotting, saturation, and rinsing, the substrates were incubated with Alexa Fluor 647-labeled streptavidin.

The binding of phosphopeptides 1P to 4P decreased as the pH value increased. At pH 6 and 7.4, weak fluorescence spots could be observed after incubation with labeled streptavidin, indicating relatively poor immobilization on the zirconium phosphonate surface (Figure 2). In contrast, much stronger binding of the four phosphopeptides was observed at pH 3 and to a lesser extent at pH 4.5. Probe spotting at pH 3 resulted in up to a 15-fold increase in fluorescence intensity compared with pH 6 and 7.4 (Figure 3 and Table S1). 1.2. SPREE (Surface Plasmon Resonance-Enhanced Ellipsometry) Experiments. To complement the array spotting experiments, binding of the peptides onto the zirconium phosphonate surface was also probed using surface plasmon resonance-enhanced ellipsometry. The poor binding affinity of the nonphosphorylated peptide (0P) was confirmed, as the

Table 1. Amino Acid Sequences Used for the Binding Study with Zirconium Phosphonate Modified Substrates name

amino acid sequences

0P 1P 2P 3P 4P

Biotin-REEDSDSDSEDE Biotin-REEDEDDD-pS-EDE Biotin-REED-pS-DDD-pS-EDE Biotin-REED-pS-DD-pS-pS-EDE Biotin-REED-pS-D-pS-pS-pS-EDE

and the influence of pH and peptide concentration during spotting was also studied. All peptides were biotinylated on the C-terminal to allow quantification of the probe immobilization upon incubation with fluorophore (Alexa Fluor 647) labeled streptavidin. Prior to incubation with labeled streptavidin, the protein α-casein, which has a high phosphate content, was used to passivate the nonspotted areas and prevent nonspecific target binding.26 Figure 2 shows an image of the spotted pattern at the end of the incubation process. It is important to note that over the whole pH range the nonphosphorylated peptide, 0P, binds very poorly to the zirconium phosphonate surface, in sharp contrast to the phosphorylated peptides (see Figure 2). Fluorescence intensities were somewhat low when using the 1 μM spotting solutions, but increased steadily for the 5 and 10 μM solutions, confirming the high density of available zirconium binding sites. The 10 μM solution consistently resulted in twice the fluorescence intensity as the 5 μM spotting solution, so subsequent experiments used the 10 μM spotting condition.

Figure 3. Fluorescence intensities as a function of pH for phosphopeptides 1P through 4P spotted from a 10 μM solution. 13951

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SPREE signal, which is proportional to the adsorbed mass, returned nearly to baseline after rinsing with buffer. On the other hand, the four phosphopeptides 1P, 2P, 3P, and 4P all exhibit specific and strong binding to the zirconium phosphonate surface at pH 3 (Figure 4). The change in

Figure 5. Fluorescence intensities quantification for non-phosphorylated proteins ((S)-affitin and (S4)-affitin) and phosphorylated protein ((S)p-affitin and (S4)p-affitin) at pH 3 and 7.4 (spotting concentration 10 μM).

approaches often lead to random protein orientation or reversible interactions.30−33 The current study explores the use of a string of phosphate groups, purposefully engineered into a protein tag, to achieve strong and directed binding of proteins to zirconated surfaces. A series of model studies using short biotinylated phosphopeptides demonstrate the feasibility of the approach, supporting the expected mechanism whereby the peptides attach to the surface via formation of zirconium phosphate coordinate covalent bonds. 1. Mechanism of Binding and Influence of pH. In the pH range studied, more acidic conditions result in enhanced phosphopeptide or phosphorylated protein binding. This result was observed in both the SPREE studies (Figure 4) and the array spotting experiments (Figures 2 and 3). The more efficient binding at low pH is consistent with the specific binding of the phosphate groups to Zr4+ ions at the zirconated surface.21,23,25−27 The phosphate groups must displace oxide/ hydroxide ligands coordinated to the highly acidic Zr4+ at the surface, which are made more labile when protonated. The point of zero charge of the zirconium-rich zirconium phosphonate surface is below pH = 3,34 so the surface charge is negative under all conditions studied. At the same time, the formal charge of the phosphopeptides is negative (Supporting Information), so in the absence of the coordinate covalent binding, the interactions are predominantly hydrogen bonding and nonspecific, with electrostatics actually opposing adsorption. The coordinate covalent binding explains why there is such a significant difference between the phosphorylated and non-phosphorylated peptides, despite the anionic charges on both. Even at pH = 7.4, where the peptide and protein immobilization is limited, those molecules that do adsorb bind strongly and are not easily rinsed away by buffer as are the nonphosphorylated peptides (Figure 2 and Figure S1). 2. Influence of the Number of Phosphate Moieties in Phosphopeptides on Their Binding Affinity. Results from the array spotting and SPREE experiments were also analyzed to investigate whether the number of serine phosphate groups in the phosphopeptide sequence has a significant influence on their affinity for the zirconium phosphonate supports. The fluorescence intensities from Figures 2 and 3 indicate that phosphopeptides 3P and 4P bind more strongly onto the surface since the fluorescence intensities are consistently 2 or 3

Figure 4. Comparison of the SPREE signal change at a zirconium phosphonate modified gold slide upon exposure to peptides 0P through 4P, 1 mg/mL, in a buffered solution at pH 3, followed by a rinsing step at pH 7.4.

SPREE signal intensity, ΔΔΨ° ≈ 0.70°−0.85°, is about the same for each phosphopeptide, indicating that approximately the same coverage is achieved no matter how many phosphate groups are present. Very little peptide is removed upon rinsing, with the 4P appearing slightly more robust than the others. In contrast, binding is significantly smaller when using buffered solutions of the peptides at pH 7.4 (Figure S1 in Supporting Information), for which the change in phase shift amplitude is only 0.25°−0.30°. These results are in good agreement with the spotting experiments and confirm that working at pH 3 results in the specific anchoring of phosphopeptides onto zirconium phosphonate surfaces while at higher pH, no significant binding occurs. 2. Protein Microarrays. The zirconium phosphonate surfaces were also used to study the immobilization of thermostable Nanofitin proteins, bearing a peptide tag containing one, (S)-affitin, or four, (S4)-affitin, phosphorylatable serine units. Specific binding via the phosphate groups was interrogated by comparing the target capture of the immobilized proteins with and without phosphorylation of the serines. After spotting of the four proteins, the zirconium phosphonate surface was blocked with α-casein and then incubated with Alexa Fluor 647-labeled lysozyme, as described in the Materials and Methods section. Two pHs were investigated, pH 3 and 7.4, and the fluorescence intensities are compared in Figure 5. Immobilization of the protein bearing four phosphate binding groups, the (S4)p-affitin, is significantly more effective than when only one phosphate function is introduced, (S)p-affitin. Also, as for the model study with the phosphorylated peptides, binding is significantly higher when the proteins are spotted at pH 3 compared to pH 7.4.

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DISCUSSION Several strategies have been developed for immobilizing proteins onto solid supports for use in biosensors, but many 13952

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times higher than for the 1P and 2P phosphopeptides. On the other hand, the binding properties of the four phosphopeptides were found to be similar in SPREE experiments. The SPREE results indicate that even one phosphate group has an affinity for the zirconated surface. An important difference between the array spotting experiments and the SPREE studies is the surface pacifying step, in this case treatment with α-casein before exposing to the fluorescent target. A phosphoprotein itself, αcasein binds strongly to the zirconium phosphonate surface to keep the fluorinated target from physisorbing to the slide and contributing to the fluorescence background. At the same time, it can compete with the adsorbed probe molecules and displace them if they are weakly bound. The passivation step appears to displace the more weakly bound 1P and to some extent 2P, but with a higher number of phosphate binding sites, the 3P and 4P peptides remain adsorbed. 3. Influence of the Position of the Peptide Phosphorylation Sites. In the microarray experiments, the fluorescence intensity of the spots from phosphopeptide 2P was slightly but consistently lower than those from phosphopeptide 1P, bringing into question whether the position of the surface attachment could influence the function of the immobilized probe. Peptides 2P, 3P, and 4P possess multiple binding phosphates, one of which is close to the biotin. This arrangement might result in the peptide being pinned to the surface close to the biotin group, potentially decreasing the mobility of the biotin and making it less available to interact with streptavidin. To test the influence of the position of phosphorylation on the peptide, a new peptide, biotinREEDEDD-pS-pS-EDE (2P′), in which the second phosphate moiety was shifted away from the biotin anchor, was prepared for comparison with 2P. Figure 6 shows the 2P vs 2P′

Figure 7. Fluorescence intensities as a function of pH for phosphopeptides (2P) and (2P′) spotted at one concentration (10 μM).

the zirconated surface was compared using SPREE (Figure 8). The two peptides lead to the same magnitude response, both

Figure 8. Comparison of the SPREE response upon adsorption of peptides 2P and 2P′ in a buffered solution (peptide concentration = 1 mg/mL) at pH 3, followed by a rinsing step at pH 7.4 and then addition of streptavidin in a buffered solution at pH 7.4, followed by a rinsing step at pH 7.4. Figure 6. Fluorescence analysis of a zirconium phosphonate modified slide spotted with phosphopeptide 2P and 2P′, at different pHs. For each condition, three spotting concentrations were used (from the left to the right: 10, 5, and 1 μM). After spotting, saturation, and rinsing, the substrates were incubated with streptavidin labeled with a fluorophore (Alexa Fluor 647).

during the adsorption step and after rinsing, indicating the same extent of surface binding. The signal change is the same as for the other peptides studied in Figure 4 under the same conditions, further suggesting that complete surface coverage is achieved when adsorbing the phosphorylated peptides. However, subsequent to the peptide binding, streptavidin was introduced to the flow cell, and in this step a difference is seen. Peptide 2P′ leads to enhanced adsorption of streptavidin, confirming differences in the capture efficiency as a function of the placement of the phosphorylated groups in the peptide chain relative to the terminal biotin. 4. Protein Binding Using a Phosphorylated Tag. From the array spotting experiments and SPREE data, we can conclude that phosphorylation of peptides makes them able to bind strongly to the zirconium phosphonate surfaces via the formation of coordinate covalent phosphate−Zr4+ bonds. To apply the idea of a phosphopeptide tag for immobilizing

comparison in an array experiment, following incubation with the fluorophore labeled streptavidin, and the measured intensities for the 10 μM spotting condition are shown in Figure 7. A significant increase in intensity is observed for 2P′ relative to 2P over the whole pH range, seemingly confirming that the biotin extremity is more available to bind streptavidin in 2P′. To confirm that the increased fluorescence in the array experiment is due to the availability of the biotin and not a result of different 2P and 2P′ surface coverage, adsorption at 13953

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Grant 0957155 (cofunded by the MPS/CHE and the Office of International Science and Engineering). The authors thank Biogenouest IMPACT academic platform, Nantes, France, for the protein microarray analysis. The Office of International Science and Engineering is also acknowledged. Finally, we are most grateful to the Genomics platform of Nantes (Biogenouest Genomics) core facility for its technical support.

proteins, segments bearing one phosphate or four phosphate moieties were fused to Nanofitin affinity proteins directed against lysozyme. A microarray study was performed to verify that the proteins modified in this manner are properly immobilized, well oriented, and still functional and capable of binding lysozyme. Based on the results obtained with the phosphopeptides, the immobilization was performed at pH 3 and 7.4, while the blocking and rinsing steps were performed at pH 7.4. The Nanofitins are naturally acidophilic, making them particularly attractive to study the effect of pH on binding, unhampered by possible denaturation.35 As shown clearly in Figure 5, when the fused tag has four phosphate binding sites, immobilization is very effective, yielding significantly more target capture than when the tag contains a single phosphate group or with no tag at all. In contrast, there is relatively little difference between binding of the nontagged protein and when the tag contains a single phosphorylation site. These observations indicate there is competition between binding at the phosphorylated tag and physisorption of the protein, so that a cluster of phosphate moieties are necessary for efficient, directed binding.

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CONCLUSION A phosphopeptide tag is shown to provide an efficient vehicle for selective and directional immobilization of proteins onto a zirconium phosphonate modified solid support. A systematic investigation shows that while a single phosphorylation site on short polypeptides will form a linkage to the surface, a cluster of phosphate groups ensures selective binding of the larger proteins, overcoming competition from nonspecific electrostatic adsorption. Given the diversity of protein structure and function and the significant dynamic range of protein−substrate affinity constants, there is room for multiple strategies for immobilizing functional proteins for use in bioanalytical applications. The binding of phosphate groups to zirconium phosphonate modified surfaces has previously been shown to be an effective strategy for immobilizing oligonucleotides27 and for supporting phospholipid membranes,36,37 and peptide tags extend the chemical strategy to proteins. In addition, the same strategy can be very likely extended to zirconium phosphonate nanoparticles, as a new tool for the selective capture of multiphosphorylated proteins in biological media, for their enrichment and further characterization.

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ASSOCIATED CONTENT

S Supporting Information *

Comparison of SPREE experiments for peptides 0P to 4P in a buffered solution at pH 7.4; calculated net charge of peptides 0P to 4P and (S4)p-affitin as a function of pH. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Maruyama, K.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Gene Expression Profiling Using DNA Microarrays. Methods Mol. Biol. 2014, 1062, 381−391. (2) Yeste, A.; Quintana, F. J. Antigen Microarrays for the Study of Autoimmune Diseases. Clin. Chem. 2013, 59, 1036−1044. (3) Berrade, L.; Garcia, A. E.; Camarero, J. A. Protein Microarrays: Novel Developments and Applications. Pharm. Res. 2011, 28, 1480− 1499. (4) Hu, S. H.; Xie, Z.; Qian, J.; Blackshaw, S.; Zhu, H. Functional Protein Microarray Technology. Wiley Interdiscip. Rev.: Syst. Biol. Med. 2011, 3, 255−268. (5) Winssinger, N.; Pianowski, Z.; Debaene, F. In Combinatorial Chemistry on Solid Supports; Brase, S., Ed.; Springer-Verlag: Berlin, 2007; Vol. 278, pp 311−342. (6) Yang, L. N.; Guo, S. J.; Li, Y.; Zhou, S. M.; Tao, S. C. Protein Microarrays for Systems Biology. Acta Biochim. Biophys. Sin. 2011, 43, 161−171. (7) Zhu, H.; Cox, E.; Qian, J. Functional Protein Microarray as Molecular Decathlete: A Versatile Player in Clinical Proteomics. Proteom. Clin. Appl. 2012, 6, 548−562. (8) Fixe, F.; Dufva, M.; Telleman, P.; Christensen, C. B. V. Functionalization of Poly(methyl methacrylate) (PMMA) as a Substrate for DNA Microarrays. Nucleic Acids Res. 2004, 32, e9. (9) Pack, S. P.; Kamisetty, N. K.; Nonogawa, M.; Devarayapalli, K. C.; Ohtani, K.; Yamada, K.; Yoshida, Y.; Kodaki, T.; Makino, K. Direct Immobilization of DNA Oligomers onto the Amine-Functionalized Glass Surface for DNA Microarray Fabrication Through the Activation-Free Reaction of Oxanine. Nucleic Acids Res. 2007, 35, e110. (10) Han, X. S. S.; He, T. Preparation and Photolithography of SelfAssembled Monolayers of 10-Mercaptodecanylphosphonic Acid on Glass Mediated by Zirconium for Protein Patterning. Colloids Surf.,B 2013, 108, 66−71. (11) Kusnezow, W.; Hoheisel, J. D. Antibody Microarrays: Promises and Problems. Biotechniques 2002, 33, S14−S23. (12) Uttamchandani, M.; Moochhala, S. Microarray-Based Enzyme Profiling: Recent Advances and Applications (Review). Biointerphases 2010, 5, FA24−FA31. (13) Janissen, R.; Oberbarnscheidt, L.; Oesterhelt, F. Optimized Straight Forward Procedure for Covalent Surface Immobilization of Different Biomolecules for Single Molecule Applications. Colloids Surf., B 2009, 71, 200−207. (14) Peluso, P.; Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Optimizing Antibody Immobilization Strategies for the Construction of Protein Microarrays. Anal. Biochem. 2003, 312, 113−124. (15) Raj, J.; Herzog, G.; Manning, M.; Volcke, C.; MacCraith, B. D.; Ballantyne, S.; Thompson, M.; Arrigan, D. W. M. Surface Immobilisation of Antibody on Cyclic Olefin Copolymer for Sandwich Immunoassay. Biosens. Bioelectron. 2009, 24, 2654−2658. (16) Katz, C.; Levy-Beladev, L.; Rotem-Bamberger, S.; Rito, T.; Rudiger, S. G. D.; Friedler, A. Studying Protein-Protein Interactions Using Peptide Arrays. Chem. Soc. Rev. 2011, 40, 2131−2145. (17) Trzcinska, R.; Balin, K.; Kubacki, J.; Marzec, M. E.; Pedrys, R.; Szade, J.; Silberring, J.; Dworak, A.; Trzebicka, B. Relevance of the Poly(ethylene glycol) Linkers in Peptide Surfaces for Proteases Assays. Langmuir 2014, 30, 5015−5025.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: clemence.queff[email protected] (C.Q.). *E-mail: [email protected]fl.edu (D.R.T.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from the “Centre National de la Recherche Scientifique” and support by the U.S. National Science Foundation Division of Chemistry under 13954

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(18) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Protein Microchips: Use for Immunoassay and Enzymatic Reactions. Anal. Biochem. 2000, 278, 123−131. (19) Gandhiraman, R. P.; Gubala, V.; O’Mahony, C. C.; Cummins, T.; Raj, J.; Eltayeb, A.; Doyle, C.; James, B.; Daniels, S.; Williams, D. E. PECVD Coatings for Functionalization of Point-of-Care Biosensor Surfaces. Vacuum 2012, 86, 547−555. (20) MacBeath, G.; Schreiber, S. L. Printing Proteins as Microarrays for High-Throughput Function Determination. Science 2000, 289, 1760−1763. (21) Cinier, M.; Petit, M.; Pecorari, F.; Talham, D. R.; Bujoli, B.; Tellier, C. Engineering of a Phosphorylatable Tag for Specific Protein Binding on Zirconium Phosphonate Based Microarrays. J. Biol. Inorg. Chem. 2012, 17, 399−407. (22) Holt, L. J.; Enever, C.; de Wildt, R. M. T.; Tomlinson, I. M. The Use of Recombinant Antibodies in Proteomics. Curr. Opin. Biotechnol. 2000, 11, 445−449. (23) Cinier, M.; Petit, M.; Williams, M. N.; Fabre, R. M.; Pecorari, F.; Talham, D. R.; Bujoli, B.; Tellier, C. Bisphosphonate Adaptors for Specific Protein Binding on Zirconium Phosphonate-based Microarrays. Bioconjugate Chem. 2009, 20, 2270−2277. (24) Lane, S. M.; Monot, J.; Petit, M.; Bujoli, B.; Talham, D. R. XPS Investigation of DNA Binding to Zirconium-Phosphonate Surfaces. Colloids Surf., B 2007, 58, 34−38. (25) Lane, S. M.; Monot, J.; Petit, M.; Tellier, C.; Bujoli, B.; Talham, D. R. Poly(dG) Spacers Lead to Increased Surface Coverage of DNA Probes: An XPS Study of Oligonucleotide Binding to Zirconium Phosphonate Modified Surfaces. Langmuir 2008, 24, 7394−7399. (26) Monot, J.; Petit, M.; Lane, S. M.; Guisle, I.; Leger, J.; Tellier, C.; Talham, D. R.; Bujoli, B. Towards Zirconium Phosphonate-Based Microarrays for Probing DNA-Protein Interactions: Critical Influence of the Location of the Probe Anchoring Groups. J. Am. Chem. Soc. 2008, 130, 6243−6251. (27) Nonglaton, G.; Benitez, I. O.; Guisle, I.; Pipelier, M.; Leger, J.; Dubreuil, D.; Tellier, C.; Talham, D. R.; Bujoli, B. New Approach to Oligonucleotide Microarrays Using Zirconium Phosphonate-Modified Surfaces. J. Am. Chem. Soc. 2004, 126, 1497−1502. (28) Massari, A. M.; Gurney, R. W.; Wightman, M. D.; Huang, C. H. K.; Nguyen, S. B. T.; Hupp, J. T. Ultrathin Micropatterned Porphyrin Films Assembled via Zirconium Phosphonate Chemistry. Polyhedron 2003, 22, 3065−3072. (29) Kretschm, E.; Raether, H. Radiative Decay of Non Radiative Surface Plasmons Excited by Light. Z. Naturforsch., A: Astrophys. Phys. Phys. Chem. 1968, A 23, 2135. (30) Jonkheijm, P.; Weinrich, D.; Schroder, H.; Niemeyer, C. M.; Waldmann, H. Chemical Strategies for Generating Protein Biochips. Angew. Chem., Int. Ed. 2008, 47, 9618−9647. (31) Lin, P. C.; Weinrich, D.; Waldmann, H. Protein Biochips: Oriented Surface Immobilization of Proteins. Macromol. Chem. Phys. 2010, 211, 136−144. (32) Rusmini, F.; Zhong, Z. Y.; Feijen, J. Protein Immobilization Strategies for Protein Biochips. Biomacromolecules 2007, 8, 1775− 1789. (33) Wong, L. S.; Khan, F.; Micklefield, J. Selective Covalent Protein Immobilization: Strategies and Applications. Chem. Rev. 2009, 109, 4025−4053. (34) Pollard, E. J. Phosphopeptide Adsorption on Zirconium Phosphate and Zirconium Phosphonate Solids. Ph.D. Thesis, University of Florida, 2013. (35) Beehar, G.; Bellinzoni, M.; Maillasson, M.; Paillard-Laurance, L.; Alzari, P. M.; He, X. M.; Mouratou, B.; Pecorari, F. Tolerance of the Archaeal Sac7d Scaffold Protein to Alternative Library Designs: Characterization of Anti-Immunoglobulin G Affitins. Protein Eng., Des. Sel. 2013, 26, 267−275. (36) Fabre, R. M.; Okeyo, G. O.; Talham, D. R. Supported Lipid Bilayers at Skeletonized Surfaces for the Study of Transmembrane Proteins. Langmuir 2012, 28, 2835−2841. (37) Fabre, R. M.; Talham, D. R. Stable Supported Lipid Bilayers on Zirconium Phosphonate Surfaces. Langmuir 2009, 25, 12644−12652. 13955

dx.doi.org/10.1021/la5036085 | Langmuir 2014, 30, 13949−13955