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Results for “Protein Array Analysis”

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At least 415 records · Page 23Linked to original sources

PubMed2004

Custom chemical microarray production and affinity fingerprinting for the S1 pocket of factor VIIa.

The goal of this study was to explore the applicability of surface plasmon resonance (SPR)-based fragment screening to identify compounds that bind to factor VIIa (FVIIa). Based on pharmacophore models virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa and immobilized these compounds on microarrays. In affinity fingerprinting experiments, a number of compounds were identified to be specifically interacting with FVIIa and shown to fall into four structural classes. The results demonstrate that the chemical microarray technology platform using SPR detection generates unique chemobiological information that is useful for de novo discovery and lead development and allows the detection of weak interactions with ligands of low molecular weight.

Chemistry, Pharmaceutical↗

PubMed2006

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip.

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.

Bacterial Proteins↗

PubMed2006

Evidence for a protective role for adiponectin in osteoarthritis.

Obesity has been associated with an increased risk of osteoarthritis (OA). However, the mechanism by which obesity contributes to OA remains uncertain. Adiponectin, an adipocyte-derived hormone, has shown anti-diabetic and anti-atherogenic properties. In the present study, we aimed to investigate the potential role of adiponectin in OA disease. We demonstrated that adiponectin was present in OA synovial fluid (SF) and its expression level was almost 100-fold decrease compared with that in OA plasma. FPLC and ELISA studies revealed the distribution and abundance of the adiponectin complexes in plasma and SF from patients with OA. The percentage of high molecular weight (HMW) per total adiponectin in OA SF was lower than in OA plasma, while that of the hexamer form was similar and the trimer form was higher. The expression levels of adiponectin receptors AdipoR1 and AdipoR2 were examined in human OA tissues by RT-PCR. AdipoR1 was abundantly expressed in cartilage, bone and synovial tissues, whereas AdipoR2 was rarely detected. Finally, the effects of adiponectin on primary chondrocyte functions were studied by using antibody-based protein array and RT-PCR. The patterns of mRNA expression and protein production strongly indicate that adiponectin is involved in the modulation of cartilage destruction in chondrocytes by up-regulating TIMP-2 and down-regulating IL-1beta-induced MMP-13. Together these findings clearly indicate that the adiponectin may act as a protective role in the progression of OA, and this also provide new thinking on the relationship between obesity and OA.

Adiponectin↗

PubMed2003

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs.

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.

Bacterial Proteins↗

PubMed2004

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array.

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.

Amino Acid Sequence↗

PubMed2004

Structural determinants of heparan sulfate interactions with Slit proteins.

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican-Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin-Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin-heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (approximately 60% at 2 microM concentration) was attained with a dodecasaccharide.

Amino Acid Sequence↗

PubMed2004

Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays.

Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.

Amino Acid Sequence↗

PubMed2006

Mapping the heparin-binding domain of human hepatic lipase.

Human hepatic lipase (HL) is known to bind to the cell surface of hepatocytes and the sinusoidal endothelium of the liver. In each case, it appears that the enzyme remains associated with the cell surface through an ionic interaction with heparan sulfate proteoglycans. However, it remains unclear as to which residues are responsible for this critical function of the enzyme. In the present study, we have used a systematic approach to map the heparin-binding regions of human HL by utilizing peptide arrays spanning the complete sequence of the mature protein. Following probing with biotin-heparin, six peptides spanning residues 301-320 and 465-476 were identified as regions binding to heparin. Probing of an additional array containing these six parent peptides and a comprehensive series of mutant peptides identified two putative HL heparin-binding domains. The first was composed of residues R310, K312, K314, and R315 at the distal N-terminal domain and the second was composed of residues R473, K474, and R476 at the C-terminal end of the protein.

Amino Acid Sequence↗

PubMed2006

Identification of proteins bound to a thioaptamer probe on a proteomics array.

A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific "on-chip" captured proteins have been obtained that were pure enough to do "on-chip" digestion and the subsequent identification of the "on-chip" bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays.

Animals↗

PubMed2006

Protein and peptide arrays: recent trends and new directions.

Microarrays of proteins and peptides make it possible the screening of thousands of binding events in a parallel and high throughput fashion; therefore they are emerging as a powerful tool for proteomics and clinical assays. The complex nature of Proteome, the wide dynamic range of protein concentration in real samples and the critical role of immobilized protein orientation must be taken into account to maximize the utility of protein microarrays. Immobilization strategy and designing of an ideal local chemical environment on the solid surface are both essential for the success of a protein microarray experiment. This review article will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications.

Combinatorial Chemistry Techniques↗

PubMed2004

Patterning protein molecules on poly(ethylene glycol) coated Si(111).

We demonstrate spatially localized immobilization of protein molecules on high-density poly(ethylene glycol) (PEG) coated Si(111). Patterns of HO- and CH3O-terminated PEG regions are formed on silicon surfaces based on soft lithography techniques and an efficient reaction between alcohol functional groups and chlorine-terminated silicon. Activation of the HO-terminated PEG brush is achieved via either partial oxidation to form aldehyde groups or via attachment of efficient leaving groups. Protein molecules are covalently immobilized to these activated regions on the PEG/Si surface.

Adsorption↗

PubMed2006

A novel glass slide-based peptide array support with high functionality resisting non-specific protein adsorption.

Glass slides have been modified with a multifunctional poly(ethylene glycol) (PEG)-based polymer with respect to array applications in the growing field of proteome research. We systematically investigated the stepwise synthesis of the PEG films starting from self-assembled alkyl silane monolayers via monolayer peroxidation and subsequent graft polymerization of PEG methacrylate (PEGMA). Chemical composition was examined by X-ray photoelectron spectroscopy (XPS); infrared spectroscopy provided information about order and composition of the films as well; film thickness was determined by ellipsometry; using fluorescence microscopy and again XPS, the amount of proteins adsorbed on the slides was investigated. The novel support material allows a versatile modification of the amino group surface density up to 40 nmol/cm(2) for the linkage of probe molecules. Further on, we carried out standard peptide synthesis based on the well-established 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, which was monitored by UV/Vis quantification of the Fmoc deblocking and mass spectrometry. The polymer coating is stable with respect to a wide range of chemical and thermal conditions, and prevents the glass surface from unspecific protein adsorption. Finally, we applied our modified glass slides in immunoassays and thus examined specific interactions of monoclonal antibodies with appropriate peptide epitopes.

Amino Acid Sequence↗

PubMed2006

Physicochemically modified silicon as a substrate for protein microarrays.

Reverse phase protein microarrays (RPMA) enable high throughput screening of posttranslational modifications of important signaling proteins within diseased cells. One limitation of protein-based molecular profiling is the lack of a PCR-like intrinsic amplification system for proteins. Enhancement of protein microarray sensitivities is an important goal, especially because many molecular targets within patient tissues are of low abundance. The ideal array substrate will have a high protein-binding affinity and low intrinsic signal. To date, nitrocellulose-coated glass has provided an effective substrate for protein binding in the microarray format when using chromogenic detection systems. As fluorescent systems, such as quantum dots, are explored as potential reporter agents, the intrinsic fluorescent properties of nitrocellulose-coated glass slides limit the ability to image microarrays for extended periods of time where increases in net sensitivity can be attained. Silicon, with low intrinsic autofluorescence, is being explored as a potential microarray surface. Native silicon has low binding potential. Through titrated reactive ion etching (RIE), varying surface areas have been created on silicon in order to enhance protein binding. Further, via chemical modification, reactive groups have been added to the surfaces for comparison of relative protein binding. Using this combinatorial method of surface roughening and surface coating, 3-aminopropyltriethoxysilane (APTES) and mercaptopropyltrimethoxysilane (MPTMS) treatments were shown to transform native silicon into a protein-binding substrate comparable to nitrocellulose.

Adsorption↗

PubMed2004

On-chip biosensing of estrogen receptor-alpha at single molecular level.

A novel method for detecting interaction between DNA and DNA-binding protein at single molecular level has been proposed. In this study, estrogen receptor-alpha (ER-alpha) was used for biosensing as the proof-example. A 518 bp-long (ca. 176 nm) DNA probe labeled with streptavidin at its 5'-terminus was prepared by inserting a consensus oligonucleotide sequence that binds to ER-alpha. A solution containing ER-alpha was dropped onto the Ni-treated mica substrate on which the DNA prove was previously immobilized, and it was observed by AFM. Specific binding of ER-alpha could be observed by measuring the distance between the site where binding occur, to the streptavidin label.

Binding Sites↗

PubMed2005

Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein.

A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.

Biosensing Techniques↗

PubMed2005

Molecularly imprinted thin film self-assembled on piezoelectric quartz crystal surface by the sol-gel process for protein recognition.

A novel method of combining sol-gel and self-assembly technology to prepare a human serum albumin (HSA)-imprinted film on the surface of piezoelectric quartz crystal (PQC) Au-electrode modified with thioglycolic acid was described in this paper. The imprinting process was characterized by using the piezoelectric quartz crystal impedance (PQCI) technique and electrochemical impedance technique. Scanning electron microscope (SEM) was employed to characterize the surface morphology of the resultant imprinted film. The piezoelectric technique and electrochemical impedance technique were also employed to investigate the binding performance of the sol-gel-imprinted film with the template protein. The results showed that the imprinted PQC film can give selective recognition to the template protein. The effects of salts and solvents on the binding capacity of the imprinted film with protein were discussed in detail. Other influencing factors (temperature and pH) have also been investigated. This self-assembly sol-gel imprinting technique was proved to be an alternative method for the preparation of biomacromolecule-imprinted thin film.

Adsorption↗

PubMed2005

Nanoparticle-assisted micropatterning of active proteins on solid substrate.

Micropatterning of proteins on silica substrate was achieved using a new method. Proteins were first immobilized onto silica nanoparticles which were then dispensed into arrayed microwells on silicon. Atomic force microscopy (AFM), fluorescence microscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the samples. The results showed that, compared to a planar surface, curved surfaces of nanoparticles provide more space for attaching proteins and thus increases the intensity of fluorescence signal. Furthermore, after attaching to silica nanoparticles, bovine serum albumin (BSA) maintains its major structure and the cytokine IFN-gamma maintains its ability to bind to its antibody. Use of this method can be extended to micropatterning of other biomolecules, such as DNA and enzymes.

Adsorption↗

PubMed2006

Lithographic techniques and surface chemistries for the fabrication of PEG-passivated protein microarrays.

This article presents a new technique to fabricate patterns of functional molecules surrounded by a coating of the inert poly(ethylene glycol) (PEG) on glass slides for applications in protein microarray technology. The chief advantages of this technique are that it is based entirely on standard lithography processes, makes use of glass slides employing surface chemistries that are standard in the microarray community, and has the potential to massively scale up the density of microarray spots. It is shown that proteins and antibodies can be made to self-assemble on the functional patterns in a microarray format, with the PEG coating acting as an effective passivating agent to prevent non-specific protein adsorption. Various standard surface chemistries such as aldehyde, epoxy and amine are explored for the functional layer, and it is conclusively demonstrated that only an amine-terminated surface satisfies all the process constraints imposed by the lithography process sequence. The effectiveness of this microarray technology is demonstrated by patterning fluorescent streptavidin and a fluorescent secondary antibody using the well-known and highly specific interaction between biotin and streptavidin.

Adsorption↗