High-throughput antibody microarrays for quantitative proteomic analysis

The antibody microarray is an intrinsically robust and quantitative system that delivers high-throughput and parallel measurements on particular sets of known proteins. It has become an important proteomics research tool, complementary to the conventional unbiased separation-based and mass spectrometry-based approaches. This review summarizes the technical aspects of production and the application for quantitative proteomic analysis with an emphasis on disease proteomics, especially the identification of biomarkers. Quality control, data analysis methods and the challenges for quantitative assays are also discussed.     

Improvement of protein stability in protein microarrays

Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml^–1 to 5 g ml^–1 by fluorescence analysis.     

Diagnostic and analytical applications of protein microarrays

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the past 5 years. A genome-scale protein microarray has been demonstrated for identifying protein–protein interactions as well as for rapid identification of protein binding to a particular drug. Furthermore, protein microarrays have been shown as an efficient tool in cancer profiling, detection of bacteria and toxins, identification of allergen reactivity and autoantibodies. They have also demonstrated the ability to measure the absolute concentration of small molecules. Besides their capacity for parallel diagnostics, microarrays can be more sensitive than traditional methods such as enzyme-linked immunosorbent assay, mass spectrometry or high-performance liquid chromatography-based assays. However, for protein and antibody arrays to be successfully introduced into diagnostics, the biochemistry of immunomicroarrays must be better characterized and simplified, they must be validated in a clinical setting and be amenable to automation or integrated into easy-to-use systems, such as micrototal analysis systems or point-of-care devices.     

Immobilization strategies for small molecule,peptide and protein microarrays

Protein, peptide and small molecule microarrays are valuable tools in biological research. In the last decade, substantial progress has been achieved to make these powerful technologies more reliable and available for researchers. This review describes chemical preparation methods for these microarrays with focus on site‐selective and bioorthogonal immobilization reactions, particularly the Staudinger ligation and the thiol‐ene reaction. In addition, the application of peptide microarrays, which were prepared by Staudinger ligation, to substrate specificity mapping is illustrated. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Microarrays of peptides elevated on the protein layer for efficient protein kinase assay

Peptide microarrays can be used for the high-throughput analysis of protein-peptide interactions. However, current peptide microarrays are rather costly to make and require cumbersome steps of introducing novel polymeric surfaces and/or chemical derivatization of peptides. Here, we report a novel method for manufacturing peptide microarrays by elevating the peptide on the layer of protein by a fusion protein approach. Using two protein kinases and their peptide substrates as examples, we show that elevating peptides on the layer of protein allows sensitive, specific, and efficient detection of peptide-protein interactions without the need for complicated chemical modification of solid supports and peptides. It was found that kinase activity could be detected with as low as 0.09 fmol of kemptide, which is about 1000-fold more sensitive than the 0.1 pmol obtained with other microarray systems. Furthermore, peptides can be produced as fusion proteins by fermentation of recombinant Escherichia coli and thus the expensive peptide synthesis process can be avoided. Therefore, this new strategy will not only be useful in high-throughput and cost-effective screening of kinase substrate peptides but also be generally applicable in studying various protein-peptide interactions.     

Signal and sensitivity enhancement through optical interference coating for DNA and protein microarray applications.

Optical inteference (OI) coated slides with unique optical properties were utilized in microarray analyses, demonstrating their enhanced detection sensitivity over traditional microarray substrates. The OI coating is comprised of a proprietary multilayered, dielectric, thin-film interference coating located beneath the functional coating (aminosilane or epoxysilane). It is designed to enhance the fluorescence in the Cy3 and Cy5 channel by increasing the light absorption of the dyes by about 6-fold and by redirecting emitted fluorescence into the detector during scanning, resulting in a theoretical limit of about 12-fold signal amplification. Two-color DNA microarray experiments conducted on the OI slides showed over 8-fold signal amplification, conservation of gene expression ratios, and increased signal-to-noise ratio when compared to control slides, indicating enhanced detection sensitivity. Protein microarray assays also exhibited over 8-fold signal amplification at three different target concentrations, demonstrating the versatility of the OI slides for different microarray applications. Further, the DNA and protein assays performed on the OI slides exhibited excellent detection sensitivity even at the low target amounts essential for diagnostic applications. The OI slides are compatible with commonly used protocols, printers, scanners and other microarray equipment. Therefore, the OI slides offer an attractive alternative to traditional microarray substrates, where enhanced detection sensitivity is desired.     

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

A protein chip approach for high-throughput antigen identification and characterization

Proteomics research in humans and other eukaryotes demands a large number of high-quality mAbs. Here, we report a new approach to produce high-quality mAbs against human liver proteins using a combined force of high-throughput mAb production and protein microarrays. After immunizing mice with live cells from human livers, we isolated 54 hybridomas with binding activities to human cells and identified the corresponding antigens for five mAbs via screening on a protein microarray of 1058 unique human liver proteins. Finally, we demonstrated that using the five mAbs we could characterize the expression profiles of their corresponding antigens by using tissue microarrays. Among them, we discovered that eIF1A expressed only in normal liver tissues, not in hepatocellular carcinoma in humans.     

Selection and characterization of peptide memitopes binding to ricin

A combinatorial random peptide display library expressed in E. coli was employed to identify short, linear peptide sequences that showed affinity for ricin and could be used as reagents for detection and identification of ricin. One peptide, P3, from a collection of four short peptides showed specific binding to ricin. The kinetic analysis of this peptide binding to the ricin showed lower equilibrium binding constants for the peptide P3 than monoclonal antibody. This is attributed due to both slower association and faster dissociation rates for the peptide P3. The random ricin peptide P3 binds to ricin with a K_D of 1 M versus the antibody's K_D of 14 nM. This particular peptide memitope P3 against ricin showed specific binding to ricin without any significant cross-reactivity against other proteins such as bovine serum albumin (BSA), lysozyme and natural bacterial toxins such as Staphylococcal enterotoxins A and B. The results provided proof-of-principal that peptide memitopes are another choice of reagents due to ease in production to be used for the detection of highly toxic bio-threat or biowarfare agents such as ricin.     

Design and validation of a neutral protein scaffold for the presentation of peptide aptamers

Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.     

Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica

Silica is widely used for chromatography resins due to its high mechanical strength, column efficiency, easy manufacturing (i.e. controlled size and porosity), and low‐cost. Despite these positive attributes to silica, it is currently used as a backbone for chromatographic resins in biotechnological downstream processing. The aim of this study is to show how the octapeptide (RH)4 can be used as peptide tag for high‐purity protein purification on bare silica. The tag possesses a high affinity to deprotonated silanol groups because the tag''s arginine groups interact with the surface via an ion pairing mechanism. A chromatographic workflow to purify GFP fused with (RH)4 could be implemented. Purities were determined by SDS‐PAGE and RP‐HPLC. The equilibrium binding capacity of the fusion protein GFP‐(RH)4 on silica is 450 mg/g and the dynamic binding capacity around 3 mg/mL. One‐step purification from clarified lysate achieved a purity of 93% and a recovery of 94%. Overloading the column enhances the purity to >95%. Static experiments with different buffers showed variability of the method making the system independent from buffer choice. Our designed peptide tag allows bare silica to be utilized in preparative chromatography for downstream bioprocessing; thus, providing a cost saving factor regarding expensive surface functionalization. Underivatized silica in combination with our (RH)4 peptide tag allows the purification of proteins, in all scales, without relying on complex resins.     

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.     

用合成多肽免疫制备尿激酶原单克隆抗体

根据尿激酶原与尿激酶一级结构的区别并结合计算机分子模拟,设计合成了包括尿激酶原Thr152-Glu163肽段的13肽,然后与载体蛋白KLH偶联作为免疫原,用BI林巴细胞融合技术获得了3种尿激酶原特异性单克隆抗体,这3种抗体仅与尿激酶原和合成多肽反应 ,而不与尿激酶及其结构类似物组织型纤溶酶原激活剂,凝血酶,纤维蛋白原反应,琼脂双向免疫扩散实验及酶活性抑制实验表明,3种抗体均为IgG类的IgG1亚类,所有3种抗体均不抑制酶活力,探讨了这组抗体用于尿激酶原结构与功能及其定量,定性分析研究方面的可能性。     

Constitution and quantity of lysis buffer alters outcome of reverse phase protein microarrays

Application of novel technology to clinical samples requires optimization of procedures. Reverse phase protein lysate arrays use femtomolar quantities of tissue lysate from clinical samples with which to profile biochemical events happening in the tumor. We analyzed the effects of different tissue solubilization buffers on frozen ovarian tumor samples in order to identify the system with the best signal intensity dynamic range, reproducibility, tissue solubility, and signal consistency. A modified RIPA-like buffer supplemented with DTT and SDS was deemed optimal.     

CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays

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In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7 × 10⁻¹³ M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.     

Peptide microarrays for the characterization of antigenic regions of human chromogranin A

Microarraying peptides is a powerful proteomics technique for studying molecular recognition events. Since peptides have small molecular mass, they are not easily accessible when adsorbed onto solid supports. Moreover, peptides can lack a well-defined three-dimensional structure, and therefore a correct orientation is essential to promote the interaction with their target. In this work, we investigated the suitability as a peptide array substrate of a glass slide coated with a copolymer of N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and [3-(methacryloyl-oxy)propyl]trimethoxysilyl. This polymeric surface was used as substrate for peptides in the characterization of linear antigenic sites of human chromogranin A, a useful tissue and serum marker for neuroendocrine tumors and a precursor of many biologically active peptides. The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which linear epitopes are freely exposed despite peptide random orientation. The results reported in this article are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.     

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.     

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.