Chemoselective surface immobilization of proteins through a cleavable peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.     

Combinations of SPR and MS for characterization of native and recombinant proteins in cell lysates

Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni²⁺ for SPR analysis of the specific Ni²⁺ binding response and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein and of cauliflower were investigated for specific binding to the phosphopeptide ligands immobilized on a sensorchip by SPR. Subsequently, the bound protein was eluted and analyzed by MS for characterization of intact mass and peptide mass mapping.     

One step immobilization of peptides and proteins by using modified parylene with formyl groups

One-step immobilization method for peptides and proteins is developed by using modified parylene film with formyl groups which is suitable for microplate-based immunoassay and SPR biosensor application. The immobilization of peptides and proteins is achieved through the covalent bonding of the formyl group with the primary amine groups of peptides and proteins, which no additional activation step is required. In this work, the immobilization efficiency of parylene-H is estimated in comparison with parylene-A and physical adsorption, using biotinylated-cyclic citrullinated peptide (biotinylated-CCP), human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) as model proteins. The applicability of parylene-H film to SPR biosensor is demonstrated by estimating the detection range and sensitivity of SPR biosensor at various thicknesses. The immobilization efficiency of parylene-H film for SPR biosensor was compared with physical adsorption by using HRP as a model protein.     

Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor

A general method to develop surface-based assays for transmembrane (TM) receptor function(s) without the need to isolate, purify, and reconstitute the proteins is presented. Based on the formation of an active surface that selectively immobilizes membrane vesicles, the method is illustrated using the chemokine receptor CCR5, a member of the largest family of cell surface eukaryotic TM proteins, the G protein-coupled receptors (GPCRs). The method begins with a protein-resistant surface containing a low percentage (1-5%) of surface-bound biotin on gold as the initial template. Surface plasmon resonance (SPR) data show specific immobilization of functional CCR5 after the initial template is activated by immobilization of rho 1D4 antibody, an anti-rhodopsin monoclonal antibody specific for the carboxyl terminal nine amino acids on bovine rhodopsin that had been engineered into the carboxyl terminus of CCR5, and exposure to vesicles obtained from mammalian cells transfected with a synthetic human CCR5 gene. Activation of the initial template is effected by sequential immobilization of avidin, which binds to the biotin in the initial template, a biotinylated goat anti-mouse immunoglobulin G (Bt-IgG), which binds to the avidin binding sites distal to the surface and the F(c) portion of the rho 1D4 antibody through its F(ab) region(s) and finally rho 1D4. This approach establishes a broad outline for the development and application of various assays for CCR5 functions. SPR data also showed that vesicle immobilization could be achieved through an integrin-integrin antibody interaction after activation of the initial template with a goat anti-human integrin beta1 antibody. These results suggest that the generic nature of the initial platform and flexibility of the subsequent surface activation for specific immobilization of membrane vesicles can be applied to the development of assays for other GPCRs or TM receptors for which antibodies are available or can be engineered to contain a particular antibody epitope.     

Antibody-antigenic peptide interactions monitored by SPR and QCM-D. A model for SPR detection of IA-2 autoantibodies in human serum

This work reports on a complementary use of surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) technologies to study interactions between a peptide antigen and polyclonal antibodies, in an experimental format suitable for diagnostic assays of autoimmune diseases. In the chosen model, a synthetic peptide from the juxtamembrane region of IA-2 (a type 1 diabetes associated antigen) was immobilized by an optimized chemical protocol applicable to both BIACORE and QCM-D sensors. A thorough study of the peptide immobilization was performed to optimize the signal-to-noise ratio using mixed self-assembled monolayers (SAM) on a gold surface. Introduction of polyethylene glycol (EG₆) chains into mixed SAM layers and addition of an anionic surfactant to the human serum reduced non-specific binding without modifying the viscoelasticity properties of the layer. Under our conditions, the antibody SPR detection limit was determined to be 0.2 nM in diluted human serum. This value is in agreement with the reported rank distribution of IA-2 antibodies in diabetic patient sera. Label-free and real-time technologies such as SPR and/or QCM-D could be precious tools in future diagnostic assays.     

Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain

Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide, G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin) and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody, and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in spite of the lower response associated with this method. K _D values of ~300 μM were determined for both strategies at pH 7.4. This compared with a K _D value of 4.4 μM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems are most suitable for future comparative screening efforts using SPR.     

Poly(HEMA) brushes emerging as a new platform for direct detection of food pathogen in milk samples

Surface plasmon resonance (SPR) biosensors capable of in real time detection of Cronobacter at concentrations down to 10? cells mL?1 in samples of consumer fresh-whole fat milk, powder whole-fat milk preparation, and powder infant formulation were developed for the first time. Antibodies against Cronobacter were covalently attached onto polymer brushes of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) grafted from the SPR chip surface. The lowest detection limit, 10? cells mL?1, was achieved in phosphate buffered saline (pH 7.4) with sensors prepared by covalent immobilization of the same antibodies onto a self assembled monolayer (SAM) of hexa(ethylene glycol) undecanethiol (EG?). However, when the EG? based sensors were challenged with milk samples the non-specific response due to the deposition of non-targeted compounds from the milk samples was much higher than the specific response to Cronobacter hampering the detection in milk. Similar interfering fouling was observed on antifouling polymer brushes of hydroxy-capped oligoethylene glycol methacrylate and even a 10 times higher fouling was observed on the widely used SAM of mixed hydroxy- and carboxy-terminated alkanethiols. Only poly(HEMA) brushes totally suppressed the fouling from milk samples. The robust well-controlled surface initiated atom transfer radical polymerization of HEMA allowed the preparation of highly dense brushes with a minimal thickness so that the capture of antigens by the antibodies immobilized on the brush layer could take place close to the gold SPR surface to provide a stronger optical response while the fouling was still suppressed. A minimum thickness of 19 nm of poly(HEMA) brush layer was necessary to suppress completely non-specific sensor response to fouling from milk.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Immobilization and clustering of structurally defined oligosaccharides for sugar chips: an improved method for surface plasmon resonance analysis of protein-carbohydrate interactions

Oligosaccharides are increasingly being recognized as important partners in receptor-ligand binding and cellular signaling. Surface plasmon resonance (SPR) is a very powerful tool for the real-time study of the specific interactions between biological molecules. We report here an advanced method for the immobilization of oligosaccharides in clustered structures for SPR and their application to the analysis of heparin-protein interactions. Reductive amination reactions and linker molecules were designed and optimized. Using mono-, tri-, or tetravalent linker compounds, we incorporated synthetic structurally defined disaccharide units of heparin and immobilized them as ligands for SPR. Their binding to an important hemostatic protein, von Willebrand factor (vWf), and its known heparin-binding domain was quantitatively analyzed. These multivalent ligand conjugates exhibited reproducible binding behavior, with consistency of the surface conditions of the SPR chip. This novel technique for oligosaccharide immobilization in SPR studies is accurate, specific, and easily applicable to both synthetic and naturally derived oligosaccharides.     

Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.     

Functional glass surface displaying a glutamyl donor substrate for transglutaminase-mediated protein immobilization

A chemically modified glass surface displaying a glutamyl donor substrate peptide (Z-QG) was developed for microbial transglutaminase (MTG)-mediated immobilization of recombinant proteins tagged with an MTG-reactive lysine-containing substrate peptide (K-tag). To evaluate the surface modification conditions affecting the enzymatic protein immobilization, we employed an amino-modified 96-well glass plate as a base and prepared three types of glass surfaces displaying Z-QG. Validation of the Z-QG modified glass surfaces with recombinant enhanced green fluorescent proteins revealed that the insertion of a di(ethylene glycol) linker between the terminal Z-QG moiety and the base not only enhances enzymatic protein immobilization efficiency but also decreases nonselective protein adsorption. A bacterial alkaline phosphatase fused with a K-tag at the N terminus was also successfully immobilized to the designed glass surface, suggesting that the chemically modified glass surface displaying a spatially controlled glutamyl donor substrate is a potential platform for MTG-mediated fabrication of protein-based solid biomaterials.     

Development of a multifunctional tool for drug screening against plasmodial protein–protein interactions via surface plasmon resonance

We have developed an expression system capable of producing large quantities of low cost, specific peptides that are either His₁₂‐tagged, biotinylated, or unlabeled. The flexibility of this peptide system is suitable for interaction studies via surface plasmon resonance (SPR), co‐crystallization, and enzyme‐linked immunosorbent assay. Gene blocks containing peptide sequences of interest in addition to a 15 amino acid AviTag?, were cloned into a vector expressing an N‐terminal maltose binding protein. The constructs were expressed and purified, and the molecular weights of the recombinant proteins were estimated by analytical size exclusion chromatography. Successful in situ biotinylation of the AviTag was confirmed by anti‐biotin western blot and was used for coupling to the surface plasmon resonance chip. We were able to validate, as a proof of concept study, the specific protein–protein interaction of Plasmodium falciparum aldolase (PfAldolase) with three different cytoplasmic adhesin tail peptides from the family of thrombospondin‐related anonymous proteins (TRAPs), and to determine their affinities. This method of peptide production enables high yield production of peptides in a two‐day, cost effective manner. This tool will allow us to screen for protein–protein interaction inhibitors directed toward the liver stage and blood stage complexes of the glideosome in Plasmodium species. Adaptation of this tool will allow researchers to pursue their own studies of protein–protein interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential binding studies applying functional protein microarrays and surface plasmon resonance

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.     

Catalytic enzyme activity on a biosensor chip: combination of surface plasmon resonance and mass spectrometry

Surface plasmon resonance (SPR) as a label-free biosensor technique has become an important tool in drug discovery campaigns during the last couple of years. For good assay performance, it is of high interest to verify the functional activity on the immobilization of the target protein on the chip. This study illustrates the verification of the catalytic activity of the drug target protein PqsD by monitoring substrate conversion as a decrease in SPR signal and product detection by ultra high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS(2)). This assay would be applicable to control surface activity of immobilized ligands.     

Optimization of DNA-directed immobilization on mixed oligo(ethylene glycol) monolayers for immunodetection

The development of protein chips has suffered from problems regarding long-term protein stability and activity. We present a protein sensor surface for immunodetection that is prepared by a DNA-directed protein immobilization method on a mixed self-assembled monolayer (SAM). By this approach, an immobilized single-stranded DNA (ssDNA) surface can be transferred/modified into a protein chip by flowing in ssDNA-conjugated protein when the protein chip measurement is needed. Therefore, the long-term stability of the protein chip will not be a problem for various applications. We tried various compositions for the SAM layer, the length of the ssDNA spacer, the end-point nucleotide composition, and the processes of ssDNA immobilization of the SAM for an optimized condition for shifting the DNA chip to a protein chip. The evaluations were made by using surface plasmon resonance. Our results indicated that a 50:1 ratio of oligo(ethylene glycol) (OEG)/COOH-terminated OEG and DNA sequences with 20mer are the best conditions found here for making a protein chip via a DNA-directed immobilization (DDI) method. The designed end-point nucleotide composition contains a few guanines or cytosines, and ssDNA immobilization of the SAM by dehybridizing immobilized double-stranded DNA (dsDNA) can improve the hybridization efficiency.     

Evaluation of surface hydrophobicity of immobilized protein with a surface plasmon resonance sensor

Immobilization is widely used to isolate agglutinative and associative proteins with large hydrophobic surfaces. Surface hydrophobicities of immobilized proteins were quantified by measuring the adsorption amounts of Triton X-100 as a hydrophobic probe with a biosensor that utilizes the phenomena of surface plasmon resonance (SPR). We measured SPR signal changes derived from adsorption of Triton X-100 to five kinds proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller equation, partly modified with a term for correcting an influence of the net charge of immobilized protein. SPR signal changes obtained by this method correlated with the values of surface hydrophobicities obtained by conventional assay using a hydrophobic probe. Thus this measuring method using an SPR sensor and Triton X-100 is expected to be a tool for quantifying surface hydrophobicities of immobilized proteins.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

Surface plasmon resonance protein sensor using Vroman effect

We report a new surface plasmon resonance (SPR) protein sensor using the Vroman effect for real-time, sensitive and selective detection of protein. The sensor relies on the competitive nature of protein adsorption onto the surface, directly depending upon protein's molecular weight. The sensor uses SPR for highly sensitive biomolecular interactions detection and the Vroman effect for highly selective detection. By using the Vroman effect we bypass having to rely on bio-receptors and their attachment to transducers, a process known to be complex and time-consuming. The protein sensor is microfabricated to perform real-time protein detection using four different proteins including aprotinin (0.65kDa), lysozyme (14.7kDa), streptavidine (53kDa), and isolectin (114kDa) on three different surfaces, namely a bare-gold surface and two others modified by OH- and COOH-terminated self-assembled monolayer (SAM). The real-time adsorption and displacement of the proteins are observed by SPR and evaluated using an atomic force microscope (AFM). The sensor can distinguish proteins of at least 14.05kDa in molecular weight and demonstrate a very low false positive rate. The protein detector can be integrated with microfluidic systems to provide extremely sensitive and selective analytical capability.