Combinatorial peptide microarrays for the rapid determination of kinase specificity

We report a rapid method for profiling of kinases using a strategy that couples the merits of combinatorics (in rapid diversity generation) with the throughput attainable using microarrays (in parallel screening). Alanine-scanning, deletion and positional-scanning peptide libraries of a kinase substrate were synthesized and site-specifically arrayed onto glass slides. The phosphorylation pattern of target sequences detected using fluorescently-labeled antiphosphoamino acid antibodies revealed the substrate preference of the kinase through its activity profile.     

Developing site-specific immobilization strategies of peptides in a microarray

In peptide-based microarrays, most existing methods do not allow for site-specific immobilization of peptides on the glass surface. We have developed two new approaches for site-specific immobilization of kinase substrates onto glass slides: (1) slides were functionalized with avidin for attachment of biotinylated peptides; and (2) slides were functionalized with thioester for attachment of N-terminally cysteine-containing peptides via a native chemical ligation reaction.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

Peptide and small molecule microarray for high throughput cell adhesion and functional assays

A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with HCl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free N(alpha)-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [gamma(33)P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the "one-bead one-compound" combinatorial library method.     

Molecular templates for bio-specific recognition by low-energy electron beam lithography

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO _x ). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO _x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.     

A chromism-based assay (CHROBA) technique for in situ detection of protein kinase activity

A unique chromism-based assay technique (CHROBA) using photochromic spiropyran-containing peptides has been firstly established for detection of protein kinase A-catalyzed phosphorylation. The alternative method has advantages that avoid isolation and/or immobilization of kinase substrates to remove excess reagents including nonreactive isotope-labeled ATP or fluorescently-labeled anti-phosphoamino acid antibodies from the reaction mixture. Such a novel protocol based on thermocoloration of the spiropyran moiety in the peptide can offer not only an efficient screening method of potent kinase substrates but also a versatile analytical tool for monitoring other post-translational modification activities.     

Ligand-modified aminobisphosphonate for linking proteins to hydroxyapatite and bone surface

An increase in bone resorption is one of the main symptoms of osteoporosis, a disease that affects more and more individuals every day. Bisphosphonates are known to inhibit bone resorption and thus are being used as a treatment for osteoporosis. Aminobisphosphonates present a functionality that can be easily used for conjugation to other molecules, such as peptides, proteins, and ligands for protein recognition. In this study, an aminobisphosphonate conjugated with biotin was used as a model linker for protein attachment to bone. With this system, the interaction of biotinylated aminobisphosphonate with hydroxyapatite, a major mineral component of bone, was investigated. Quantification of the binding of aminobisphosphonate to hydroxyapatite was performed using a fluorescently labeled antibody for biotin. Additionally, the interaction of the biotinylated aminobisphosphonate with multiple treatments of cortical bone from the midshaft of a cow femur was studied. It was demonstrated that modified aminobisphosphonate reagents can bind hydroxyapatite and bone at high levels, while the biotin functionality is free to be recognized by the fluorescently labeled antibiotin antibody, suggesting that modified aminobisphosphonates could be used to link other peptides or proteins to the bone surface.     

Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays

A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way.     

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.     

Enhancing bioplastic-substrate interaction via pore induction and directed migration of enzyme location

We demonstrate two novel approaches to enhance interactions of polymer-immobilized biomolecules with their substrates. In the first approach, diisopropylfluorophosphatase (DFPase) containing poly(urethane) (PU) coatings were made microporous by incorporating, then extracting, poly(ethylene glycol)-based diesters as porogens. Incorporation of 2% w/w porogen increased the effective diffusion coefficient of diisopropylfluorophosphate (DFP) through the coatings by 30% and increased the apparent turnover number of immobilized DFPase 3-fold. In the second approach, prior to immobilization, hydrophobic modification of DFPase was achieved through its conjugation with a dimer/trimer mixture of a uretdione based on 1,6-diisocyanatohexane. When the hydrophobically modified DFPase was immobilized in coatings, catalytic activity was 4-fold higher than that of the equivalent, immobilized, native DFPase. This activity enhancement was independent of the presence or absence of pores. Confocal microscopy images of coatings containing fluorescently labeled lysozyme show that the native enzyme is distributed uniformly over the entire thickness of the coatings. Hydrophobically modified and fluorescently labeled lysozyme is accumulated only in the upper 10 microm cross-sectional layer of a 100 microm-thick coating. Interactions of bioplastics with their substrates are tunable either by pore induction in a polymer or by directed migration of the hydrophobically modified biomolecule to the desired location. The latter approach has broad implications, including overcoming mass transfer limitations experienced by immobilized biocatalysts.     

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.     

Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV

Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.     

An Activated GOPS‐poly‐L‐Lysine‐ Coated Glass Surface for the Immobilization of 60mer Oligonucleotides

To explore a method for enhancing the immobilization and hybridization efficiency of oligonucleotides on DNA microarrays, conventional protocols of poly‐L‐lysine coating were modified by means of surface chemistry, namely, the slides were prepared by the covalently coupling of poly‐L‐lysine to a glycidoxy‐modified glass surface. The modified slides were then used to print microarrays for the detection of the SARS coronavirus by means of 60mer oligonucleotide probes. The characteristics of the modified slides concerning immobilization efficiency, hybridization dynamics, and probe stripping cycles were determined. The improved surface exhibited high immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics. The spotting concentration of 10 μmol/L can meet the requirements of detection; the spots were approximately 170 nm in diameter; the mean fluorescence intensity of the SARS spots were between 3.2 × 10⁴ and 5.0 × 10⁴ after hybridization. Furthermore, the microarrays prepared by this method demonstrated more resistance to consecutive probe stripping cycles. The activated GOPS‐PLL slide could undergo hybridization stripping cycles for at least three cycles, and the highest loss in fluorescence intensity was found to be only 11.9 % after the third hybridization. The modified slides using the above‐mentioned method were superior to those slides treated with conventional approaches, which theoretically agrees with the fact that modification by surface chemistry attaches the DNA covalently firmly to the slides. This protocol may have great promise in the future for application in large‐scale manufacture.     

Efficacy of the phosphorylation of synthetic peptides by purified catalytic subunit of PKA (PKAcat) from bovine lens depends on the amino acid sequence of the peptides.

Protein kinase (PK) A catalytic (PKAcat) subunit was purified to homogeneity from bovine lens using a 100-kDa cut-off membrane filtration followed by different chromatographic procedures. The molecular weight of PKAcat was found to be 41 kDa. The kinase phosphorylates histone IIIs and other synthetic modified peptides of VRKRTLRRL with different amino acid environment. The extent of phosphorylation depends not only on the presence of Ser or Thr (phosphorylating residues) but also on other surrounding amino acid residues. Although some peptides compete in phosphorylating histone, they are not very significant. The result suggests that the extent of phosphorylation depends on the amino acid residue(s) surrounding phosphorylable residue(s) on the peptide.     

An antibody to a synthetic peptide recognizes polyomavirus middle-T antigen and reveals multiple in vitro tyrosine phosphorylation sites.

Antibodies were raised against three synthetic peptides corresponding to sequences surrounding tyrosine 315, a putative in vitro phosphorylation site in polyomavirus middle-T antigen. Only one of the peptides (called C and corresponding to residues 311 to 330) elicited antibodies that recognized middle-T efficiently. Middle-T present in immunoprecipitates formed with purified anti-C serum still accepted phosphate on tyrosine in an in vitro kinase reaction. This implies that tyrosines other than 315 and 322 that lie within the antibody binding region are phosphorylated under these conditions. This conclusion was supported by the altered partial V8 proteolysis fingerprint of the labeled middle-T. Two-dimensional tryptic fingerprint analysis of 32P-labeled middle-T showed that several tryptic peptides identified as including tyrosine 315 and 322 were missing from middle-T labeled in anti-C immunoprecipitates compared with middle-T labeled in immunoprecipitates made by using anti-tumor cell serum. However, one major labeled peptide remained. This peptide was also present in fingerprints of 32P-labeled middle-T coded by M45, dl23, pAS131, and dl1013, but a peptide with altered mobility was present in dl8 middle-T. This identified the peptide as including tyrosine 250. We deduce from these data that (i) the presence of the antibody against peptide C inhibits phosphorylation of tyrosines 315 and 322; (ii) middle-T labeled in the kinase reaction after immunoprecipitation with anti-C serum is phosphorylated on tyrosine 250; and (iii) when anti-tumor cell serum is used in the in vitro kinase reaction, middle-T is phosphorylated at multiple sites, including residues 250, 315, and 322.     

Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles

We used fluorescence correlation spectroscopy (FCS) to analyze the binding of fluorescently labeled peptides to lipid vesicles and compared the deduced binding constants to those obtained using other techniques. We used a well-characterized peptide corresponding to the basic effector domain of myristoylated alanine-rich C kinase substrate, MARCKS(151-175), that was fluorescently labeled with Alexa488, and measured its binding to large unilamellar vesicles (diameter approximately 100 nm) composed of phosphatidylcholine and phosphatidylserine or phosphatidylinositol 4,5-bisphosphate. Because the large unilamellar vesicles are significantly larger than the peptide, the correlation times for the free and bound peptide could be distinguished using single color autocorrelation measurements. The molar partition coefficients calculated from the FCS measurements were comparable to those obtained from binding measurements of radioactively labeled MARCKS(151-175) using a centrifugation technique. Moreover, FCS can measure binding of peptides present at very low concentrations (1-10 nmolar), which is difficult or impossible with most other techniques. Our data indicate FCS can be an accurate and valuable tool for studying the interaction of peptides and proteins with lipid membranes.     

Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays

The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Anti-KDEL-coated nanoparticles: A promising tumor targeting approach for ovarian cancer?

The purpose of this study was to target ovarian cancer cells by coupling paclitaxel (Tx)-loaded nanoparticles (NPs-Tx) to antibodies against KDEL sequence, able to recognize GRP94 and GRP78 that are located at cell surface in cancer cells whereas they are in the endoplasmic reticulum in healthy cells. Tx-loaded poly (dl-lactic acid) nanoparticles coated with anti-KDEL antibodies (NPs-Tx-KDEL) were successfully prepared and characterized. Interaction between tumor cells and NPs-Tx or NPs-Tx-KDEL was observed by microscopy with fluorescently labeled NPs and the efficacy of the different formulations was compared by a viability assay.     

Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations

Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphorylation of rhodopsin is about 10(3) lower than that for the peptides, which suggests that binding of rhodopsin kinase to its substrate, photolyzed rhodopsin, involves more than just binding to the carboxyl-terminal peptide region that is to be phosphorylated. A synthetic peptide from the rhodopsin sequence that contains both serines and threonines is improved as a substrate by substitution of serines for the threonines, suggesting that serine residues are preferred as substrates. Analogous 25 amino acid peptides from the human red or green cone visual pigment, a beta-adrenergic receptor, or M1 muscarinic acetylcholine receptors are better substrates for bovine rhodopsin kinase than is the peptide from bovine rhodopsin. An acidic serine-containing peptide from a non-receptor protein, alpha s1B-casein, is also a good substrate for rhodopsin kinase. However, many basic peptides that are substrates for other protein kinases--histone IIA, histone IIS, clupeine, salmine, and a neurofilament peptide--are not phosphorylated by rhodopsin kinase. Polycations such as spermine or spermidine are nonessential activators of phosphorylation of rhodopsin or its synthetic peptide 324-348. Polyanions such as poly(aspartic acid), dextran sulfate, or poly(adenylic acid) inhibit the kinase. Poly(L-aspartic acid) is a competitive inhibitor with respect to rhodopsin (KI = 300 microM) and shows mixed type inhibition with respect to ATP.