Protein–DNA Arrays as Tools for Detection of Protein–Protein Interactions by Mass Spectrometry

Analysis of multiple protein–protein interactions using microarray technology remains challenging, and site‐specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein–DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt–DNA hybrid molecules described here are used for DNA‐directed immobilization on glass‐ and silica‐based microarrays. Methods for the detection of protein–DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP‐ and GTP‐bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt‐effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.     

Lipochitin Oligosaccharides Immobilized through Oximes in Glycan Microarrays Bind LysM Proteins

Glycan microarrays have emerged as novel tools to study carbohydrate–protein interactions. Here we describe the preparation of a covalent microarray with lipochitin oligosaccharides and its use in studying proteins containing LysM domains. The glycan microarray was assembled from glycoconjugates that were synthesized by using recently developed bifunctional chemoselective aminooxy reagents without the need for transient carbohydrate protecting groups. We describe for the first time the preparation of a covalent microarray with lipochitin oligosaccharides and its use for studying proteins containing LysM domains. Lipochitin oligosaccharides (also referred to as Nod factors) were isolated from bacterial strains or chemoenzymatically synthesized. The glycan microarray also included peptidoglycan‐related compounds, as well as chitin oligosaccharides of different lengths. In total, 30 ligands were treated with the aminooxy linker molecule. The identity of the glycoconjugates was verified by mass spectrometry, and they were then immobilized on the array. The presence of the glycoconjugates on the array surface was confirmed by use of lectins and human sera (IgG binding). The functionality of our array was tested with a bacterial LysM domain‐containing protein, autolysin p60, which is known to act on the bacterial cell wall peptidoglycan. P60 showed specific binding to Nod factors and to chitin oligosaccharides. Increasing affinity was observed with increasing chitin oligomer length.     

Preparation of protein microarrays on non‐fouling and hydrated poly(ethylene glycol) hydrogel substrates using photochemical surface modification

BACKGROUND: Conventional protein microarrays prepared on hard, dry substrates, such as glass and silicone, have several limitations, as proteins may easily denature and lose their structure. To overcome such problems, the fabrication of wet protein microarrays on non‐fouling and hydrated PEG‐based hydrogels was investigated. RESULT: Bovine serum albumin (BSA) and glucose oxidase (GOX), chosen as model proteins, were covalently immobilized on PEG hydrogel surfaces via 5‐azidonitrobenzoyloxy N‐hydroxysuccinimide, a photoreactive bifunctional linker. Successful fixation of the bifunctional linker and subsequent immobilization of the proteins on the PEG hydrogel surfaces were confirmed with X‐ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) studies. GOX immobilized on the hydrogel surface maintained approximately 50% of its initial activity after 24 h when left in dry conditions, but maintained only 20% when immobilized on a dry substrate. Photochemical fixation combined with photolithography produced well‐defined protein micropatterns with sizes ranging from 50–500 µm, and molecular recognition‐mediated specific binding between biotin and streptavidin was successfully assayed using microarrays on PEG hydrogels. CONCLUSION: A protein‐repellent PEG hydrogel surface was photochemically modified to covalently immobilize proteins and create protein microarrays. The use of hydrated hydrogels as substrates for protein microarrays could minimize the deactivation of proteins in dry conditions, and the non‐fouling property of PEG hydrogels allows the passivation step of protein microarray preparation to be skipped. Copyright © 2008 Society of Chemical Industry     

Rapid Screening of Lectins for Multivalency Effects with a Glycodendrimer Microarray

Multivalency is an important phenomenon in protein–carbohydrate interactions. In order to evaluate glycodendrimers as multivalent inhibitors of carbohydrate binding proteins, we displayed them on a microarray surface. Valencies were varied from 1 to 8, and corrections were made for the valencies so that all surfaces contained the same amount of the sugar ligand. Five different carbohydrates were attached to the dendrimers. A series of fluorescent lectins was evaluated, and for each of them a binding profile was obtained from a single experiment showing both the specificity of the lectin for a certain sugar and whether it prefers multivalent ligands or not. Very distinct binding patterns were seen for the various lectins. The results were rationalized with respect to the interbinding distances of the lectins.     

Characterization of Optimal Aptamer‐Microarray Binding Chemistry and Spacer Design

Compared to conventional protein microarrays, the aptamer microarray is a relatively new and competitive method for analytical applications. The three‐dimensional folded aptamers show excellent binding specificity and affinity, and thus, can be used as an alternative to antibodies. Immobilization and binding effects of aptamers under different combinations of surfaces and spacers for aptamer‐microarray applications are investigated. In addition, the effects of spacers integrated at the terminal position of aptamers on made in‐house and commercially available microarray supports are explored.     

Effects of the Surface Densities of Glycoclusters on the Determination of Their IC50 and Kd Value Determination by Using a Microarray

Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10–30 % of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health. One promising therapeutic option is to design glycoclusters that target the virulence factor of PA. LecA is a galactose‐specific lectin that might be involved in adhesion and biofilm formation by PA. The DNA‐directed immobilization (DDI) microarray is a powerful tool for screening and understanding of structure–activity relationships between glycoclusters and lectins. High‐throughput and multiplexed analysis of lectin–glycocluster interactions on a DDI microarray allows measurement of IC₅₀ and dissociation constant (K_d) values with minute amounts of material. In order to study the robustness of the DDI microarray in determination of IC₅₀ and K_d values, the impact of glycocluster surface density was investigated. The data obtained show that measured IC₅₀ values were influenced by glycocluster surface density: as the density of glycoclusters increases, the measured IC₅₀ values increase too. In contrast, the measured K_d values were not affected by glycocluster surface density, provided that the experimental conditions allow interaction between glycocluster and lectin at single‐molecule level (no surface cluster effect).     

A simple and cost-effective method to prepare template DNAs for cell-free protein synthesis

The scale-up of cell-free protein synthesis reactions involves the preparation of large amounts of template DNA. While ion-exchange column chromatography methods have commonly been used to obtain purified plasmid DNA for cell-free protein synthesis reactions, these methods are costly and difficult to expand to a large scale. In this work, we report that the routine isopropyl alcohol (IPA) precipitation method can be used to prepare cell-free-expressible DNA when the co-precipitated proteins are removed. Compared to column-purification procedures, the IPA-precipitation offers obvious advantages with respect to the cost and scaling-up of template preparation, and we believe that our finding will contribute to making cell-free protein synthesis system more practical for the rapid production of preparative amounts of recombinant proteins.     

Protein Functionalized Nanodiamond Arrays

Various nanoscale elements are currently being explored for bio-applications, such as in bio-images, bio-detection, and bio-sensors. Among them, nanodiamonds possess remarkable features such as low bio-cytotoxicity, good optical property in fluorescent and Raman spectra, and good photostability for bio-applications. In this work, we devise techniques to position functionalized nanodiamonds on self-assembled monolayer (SAMs) arrays adsorbed on silicon and ITO substrates surface using electron beam lithography techniques. The nanodiamond arrays were functionalized with lysozyme to target a certain biomolecule or protein specifically. The optical properties of the nanodiamond-protein complex arrays were characterized by a high throughput confocal microscope. The synthesized nanodiamond-lysozyme complex arrays were found to still retain their functionality in interacting with E. coli.     

Dansyl,a fluorescent photoprotecting group for microarray applications

BACKGROUND: The fluorescence dye 5‐dimethylamino‐1‐naphthalenesulfonyl chloride (Dansyl chloride) is commonly used for labeling the N‐terminus of proteins and peptides. Apart from the fluorescence, the ? SO₂? NH? bonds formed are susceptible to photolytic cleavage and will subsequently restore free amines. Consequently, Dansyl amides could act as a fluorescent photoprotecting group with novel application in solid phase synthesis or in microarray technologies. RESULTS: Commercial microscope glass slides were silanized with (3‐aminopropyl) triethoxysilane, exposed amines were activated with 1,4‐phenylene diisothiocyanate and subsequently reacted with dansylated polyethylene imine (PEI). The resulting fluorescence of the surface was determined and used as a measure of the homogeneity of the introduced functional groups. Using a mask, Dansyl‐PEI modified slides were locally exposed to photolytic cleavage within irradiation energy of 100 J cm^?2. Inscribed structures would be easily recognized due to their loss of fluorescence. The restored amines in deprotected areas were reacted with phosphorylated capture oligonucleotides followed by hybridization with complementary Cy5‐labeled targets. CONCLUSIONS: Capture probes immobilized precisely in structures exposed to UV‐light while non‐irradiated areas remained blocked. Such pre‐structured surfaces allow the production of highly reproducible microarrays without any specific problems of spotting imperfections. Gridding and segmentation of the determined sample allocation facilitate spot finding and spot analysis. Copyright © 2012 Society of Chemical Industry     

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.     

Chromophore of an Enhanced Green Fluorescent Protein Can Play a Photoprotective Role Due to Photobleaching

Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO₂, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.     

DNA‐Templated Introduction of an Aldehyde Handle in Proteins

Many medical and biotechnological applications rely on protein labeling, but a key challenge is the production of homogeneous and site‐specific conjugates. This can rarely be achieved by simple residue‐specific random labeling, but generally requires genetic engineering. Using site‐selective DNA‐templated reductive amination, we created DNA–protein conjugates with control over labeling stoichiometry and without genetic engineering. A guiding DNA strand with a metal‐binding functionality facilitates site‐selectivity by directing the coupling of a second reactive DNA strand in the vicinity of a protein metal‐binding site. We demonstrate DNA‐templated reductive amination for His₆‐tagged proteins and metal‐binding proteins, including IgG1 antibodies. We also used a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde on the protein. This functions as a handle for further modifications with desired labels. In addition to directing the aldehyde positioning, the DNA provides a straightforward route for purification between reaction steps.     

Systematical Screening of Intracellular Protein Targets of Polyphemusin-I Using Escherichia coli Proteome Microarrays

With their wide repertoire of mechanisms, antimicrobial peptides (AMPs) are promising alternatives to fight against varied pathogenic microorganisms (bacteria, fungi, viruses, parasites, etc.). AMPs, novel components of the innate immune defense system, are secreted by all organisms. The aquatic environment represents a huge population and an enormous source of varied AMPs. Polyphemusin-I, a marine AMP isolated from hemocytes of an American horseshoe crab, possesses high antimicrobial activities. Studies on polyphemusin-I have verified the intracellular mechanisms of action, however, its intracellular targets are not yet explored. In this study, we employed Escherichia coli proteome microarrays to systematically screen the entire intracellular protein targets of polyphemusin-I. A total of 97 protein targets of polyphemusin-I were statistically analyzed from the quadruplicate Escherichia coli proteome microarrays assays. Among these identified protein targets, 56 proteins had cellular location inside the cell (i.e., cytoplasm), one in the plasma membrane, one in the periplasm and the rest 39 proteins had no specified cellular location. The bioinformatics analysis of these identified protein targets of polyphemusin-I in gene ontology (GO) enrichment category of molecular function revealed significant enrichment in nucleic acid related GO terms i.e., “RNA binding”, “nucleotide binding”, “nuclease activities”, “uracil DNA N-glycosylase activities” and others. Moreover, enrichment in GO category of biological process also depicted enrichment in nucleic acid related GO terms, such as “nucleic acid phosphodiester bond hydrolysis”, “deoxyribonucleotide metabolism”, and others. In accordance to GO enrichment analysis, protein families (PFAM) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis also showed significant enrichment in nucleic acid terms. These enrichment results suggest that polyphemusin-I targets nucleic acid-associated proteins. Furthermore, to provide a comprehensive study, we compared the identified protein targets of polyphemusin-I with previously identified protein targets of four AMPs (P-Der, Lfcin B, PR-39, and Bac 7) using Escherichia coli proteome microarrays. The comparison study of five AMPs (polyhemusin-I, P-Der, Lfcin B, PR-39, and Bac 7) showed only nine common protein targets in all the five AMPs, whereas a total of 39 and 43 common protein targets were identified among the two marine AMPs (polyphemusin-I and P-Der) and three terrestrial AMPs (Lfcin B, PR-39 and Bac7), respectively. To further reveal the target pattern of marine and terrestrial AMPs, the enrichment results obtained from common protein targets of marine AMPs with terrestrial AMPs were compared. The comparison result indicated that AMPs have unique mechanism of action among marine or terrestrial AMPs. Hence, in this study, we have not only identified the intracellular protein targets of polyphemusin-I, but also revealed the protein target differences between marine AMPs and terrestrial AMPs.     

Photoradical-Mediated Catalyst-Independent Protein Cross-Link with Unusual Fluorescence Properties

Photo-actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both in vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site-selective incorporation of 7-fluoro-indole in the human mitochondrial outer membrane protein VDAC2 (voltage-dependent anion channel isoform 2), with the aim of generating Trp−Phe/Tyr cross-links. Prolonged irradiation at 282 nm provided us with a surprisingly unusual fluorophore that displayed sizably red-shifted excitation (λ_ex-max=280 nm→360 nm) and emission (λ_em-max=330 nm→430 nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo-activated cross-linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site-specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein-independent. Our findings reveal the photoradical-mediated accumulation of reversible tyrosine cross-links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV-mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.     

Protein-detecting microarrays: current accomplishments and requirements

The sequencing of the human genome has been successfully completed and offers the chance of obtaining a large amount of valuable information for understanding complex cellular events simply and rapidly in a single experiment. Interestingly, in addressing these proteomic studies, the importance of protein-detecting microarray technology is increasing. In the coming few years, microarray technology will become a significantly promising and indispensable research/diagnostic tool from just a speculative technology. It is clear that the protein-detecting microarray is supported by three independent but strongly related technologies (surface chemistry, detection methods, and capture agents). Firstly, a variety of surface-modification methodologies are now widely available and offer site-specific immobilization of capture agents onto surfaces in such a way as to keep the native conformation and activity. Secondly, sensitive and parallel detection apparatuses are being developed to provide highly engineered microarray platforms for simultaneous data acquisition. Lastly, in the development of capture agents, antibodies are now probably the most prominent capture agents for analyzing protein abundances. Alternative scaffolds, such as phage-displayed antibody and protein fragments, which provide the advantage of increasing diversity of proteinic capture agents, however, are under development. An approach involving recombinant proteins fused with affinity tag(s) and coupled with a highly engineered surface chemistry will provide simple production protocols and specific orientations of capture agents on the microarray formats. Peptides and other small molecules can be employed in screening highly potent ligands as well as in measuring enzymatic activities. Protein-detecting microarrays supported by the three key technologies should contribute in accelerating diagnostic/biological research and drug discovery.     

Dendrimer-activated solid supports for nucleic acid and protein microarrays

The generation of chemically activated glass surfaces is of increasing interest for the production of microarrays containing DNA, proteins, and low-molecular-weight components. We here report on a novel surface chemistry for highly efficient activation of glass slides. Our method is based on the initial modification of glass with primary amino groups using a protocol, specifically optimized for high aminosilylation yields, and in particular, for homogeneous surface coverages. In a following step the surface amino groups are activated with a homobifunctional linker, such as disuccinimidylglutarate (DSG) or 1,4-phenylenediisothiocyanate (PDITC), and then allowed to react with a starburst dendrimer that contains 64 primary amino groups in its outer sphere. Subsequently, the dendritic monomers are activated and crosslinked with a homobifunctional spacer, either DSG or PDITC. This leads to the formation of a thin, chemically reactive polymer film, covalently affixed to the glass substrate, which can directly be used for the covalent attachment of amino-modified components, such as oligonucleotides. The resulting DNA microarrays were studied by means of nucleic acid hybridization experiments using fluorophor-labeled complementary oligonucleotide targets. The results indicate that the novel dendrimer-activated surfaces display a surface coverage with capture oligomers about twofold greater than that with conventional microarrays containing linear chemical linkers. In addition, the experiments suggest that the hybridization occurs with decreased steric hindrance, likely a consequence of the long, flexible linker chain between the surface and the DNA oligomer. The surfaces were found to be resistant against repeated alkaline regeneration procedures, which is likely a consequence of the crosslinked polymeric structure of the dendrimer film. The high stability allows multiple hybridization experiments without significant loss of signal intensity. The versatility of the dendrimer surfaces is also demonstrated by the covalent immobilization of streptavidin as a model protein.     

The Ionic Modification of the Surface Charge and Isoelectric Point of Soy Protein

The effect of anionic and cationic binding on the surface charge of soy proteins was measured by electrokinetic analysis. All of the ions investigated suppressed the surface charge of the protein; however, certain multivalent ions such as Al (III), Fe (III), hexametaphosphate and tripolyphosphate also altered the isoelectric point of the protein. The results indicated the unpredictability of ionic effects on protein functionality, thus emphasizing the importance of making measurements of protein charge.     

Synthesis and investigation of a new macroporous monolithic material based on an N‐hydroxyphthalimide ester of acrylic acid‐co‐glycidyl methacrylate‐co‐ethylene dimethacrylate terpolymer

A macroporous monolithic material based on an N‐hydroxyphthalimide ester of acrylic acid‐co‐glycidyl methacrylate‐co‐ethylene dimethacrylate terpolymer was synthesized by photoinitiated free‐radical polymerization. Several porogenic solvents, such as cyclohexanol, dodecanol, and poly(ethylene glycol)s, were tested to obtain the monolithic material with an optimal pore size allowing unrestricted penetration of large molecule (proteins) into a three‐dimensional porous space. The new monolithic material was covalently bound to an inert surface (glass) directly in the polymerization step, and it was suggested as a solid matrix for the development of new types of three‐dimensional protein microarrays (biochips). A demonstration of the potential of the suggested microarray platform as well as optimization of microarray performance conditions was realized with a model mouse immunoglobulin G/goat anti‐mouse immunoglobulin G affinity pair. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Peroxodisulfate generation on boron-doped diamond microelectrodes array and detection by scanning electrochemical microscopy

In this work, the electrogeneration of peroxodisulfate from a 1 M H₂SO₄ solution on boron-doped diamond microelectrodes array has been studied. The peroxodisulfate is detected at the vicinity of the boron doped diamond electrode, with the SECM probe, only when the polarization of the microarray is greater than 2.1 V versus AgCl/Ag. The main electrochemical interest of working with microarrays comes from the fact that current densities and efficiencies are comparable to those observed in the peroxodisulfate industrial production. The local Scanning Electrochemical Microscopy studies have shown that the peroxodisulfate reaction is related to a surface mediated oxidation of sulfate anions into peroxodisulfate according to a complex reactional mechanism.