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At least 577 records · Page 32Linked to original sources
PubMed2004

Micro patterning of cell and protein non-adhesive plasma polymerized coatings for biochip applications.

Micro scale patterning of bioactive surfaces is desirable for numerous biochip applications. Polyethyleneoxide-like (PEO-like) coating with non-fouling functionality has been deposited using low frequency AC plasma polymerization. The non-fouling properties of the coating were tested with human cells (HeLa) and fluorescence labeled proteins (isothiocyanate-labeled bovine serum albumin, i.e. FITC-BSA). The PEO-like coatings were fabricated by plasma polymerization of 12-crown-4 (ppCrown) with plasma polymerized hexene (ppHexene) as adhesion layer. The coatings were micro patterned using conventional cleanroom photolithography and lift-off. Single cell arrays showed sharp contrast in cell adhesion between the untreated glass surface and the ppCrown layer. Similarly, proteins adsorbed selectively to untreated glass but not to ppCrown. The simplicity of the lift-off technique and the sturdiness and versatility of the plasma-polymerized coatings, make this technology highly suitable for bio-MEMS and biochip applications, where patterned high contrast non-fouling surfaces are needed.

Adsorption↗
PubMed2005

Fast and reliable protein microarray production by a new drop-in-drop technique.

In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.

Animals↗
PubMed2005

Writing 3D protein nanopatterns onto a silicon nanosponge.

A three-dimensional protein nanopatterning method has been developed, based on local activation of porous silicon by electron beam. Proteins specifically bind to irradiated regions, and the depth of biomolecule nanopatterns can be controlled by varying the electron energy. This unique feature permits exploitation of the huge surface area of the sponge-like material, thus allowing concentration of a large amount of proteins on nanosized patterns. Moreover, the grafted biomolecules retain their full functionality, and the feasibility of a glucose sensor has been demonstrated.

Cytoplasm↗
PubMed2006

Zeolite nanoparticle modified microchip reactor for efficient protein digestion.

An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol-gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon-oxygen-silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s(-1). Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL min(-1), which affords a very short reaction time of less than 5 s.

Amino Acid Sequence↗
PubMed2006

Analytical platforms for activity-based protein profiling--exploiting the versatility of chemistry for functional proteomics.

The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.

Animals↗
PubMed2006

Proteomics technology in systems biology.

It has now become apparent that a full understanding of a biological process (e.g. a disease state) is only possible if all biomolecular interactions are taken into account. Systems biology works towards understanding the intricacies of cellular life through the collaborative efforts of biologists, chemists, mathematicians and computer scientists and recently, a number of laboratories around the world have embarked upon such research agendas. The fields of genomics and proteomics are foundational in systems biology studies and a great deal of research is currently being conducted in each worldwide. Moreover, many technological advances (particularly in mass spectrometry) have led to a dramatic rise in the number of proteomic studies over the past two decades. This short review summarizes a selection of technological innovations in proteomics that contribute to systems biology studies.

Chromatography, Affinity↗
PubMed2004

Enthalpy arrays.

We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.

2,4-Dinitrophenol↗
PubMed2004

Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips.

The TOR (target of rapamycin) proteins play important roles in nutrient signaling in eukaryotic cells. Rapamycin treatment induces a state reminiscent of the nutrient starvation response, often resulting in growth inhibition. Using a chemical genetic modifier screen, we identified two classes of small molecules, small-molecule inhibitors of rapamycin (SMIRs) and small-molecule enhancers of rapamycin (SMERs), that suppress and augment, respectively, rapamycin's effect in the yeast Saccharomyces cerevisiae. Probing proteome chips with biotinylated SMIRs revealed putative intracellular target proteins, including Tep1p, a homolog of the mammalian PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor, and Ybr077cp (Nir1p), a protein of previously unknown function that we show to be a component of the TOR signaling network. Both SMIR target proteins are associated with PI(3,4)P2, suggesting a mechanism of regulation of the TOR pathway involving phosphatidylinositides. Our results illustrate the combined use of chemical genetics and proteomics in biological discovery and map a path for creating useful therapeutics for treating human diseases involving the TOR pathway, such as diabetes and cancer.

Humans↗
PubMed2004

Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.

Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.

Carbohydrate Sequence↗
PubMed2005

P2Y1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2.

P2Y(1) purinergic receptors (P2Y(1)Rs) mediate rises in intracellular Ca(2+) in response to ATP, but the duration and characteristics of this Ca(2+) response are known to vary markedly in distinct cell types. We screened the P2Y(1)R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95/Drosophila Discs large/ZO-1 homology) domains and identified a previously unrecognized, specific interaction with the second PDZ domain of the scaffold NHERF-2 (Na(+)/H(+) exchanger regulatory factor type 2). Furthermore, we found that P2Y(1)R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y(1)R to key downstream effectors such as phospholipase Cbeta. Finally, we found that coexpression of P2Y(1)R with NHERF-2 in glial cells prolongs P2Y(1)R-mediated Ca(2+) signaling, whereas disruption of the P2Y(1)R-NHERF-2 interaction by point mutations attenuates the duration of P2Y(1)R-mediated Ca(2+) responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y(1)R and may therefore determine cell-specific differences in P2Y(1)R-mediated signaling.

Blotting, Western↗
PubMed2005

Protein "fingerprinting" in complex mixtures with peptoid microarrays.

We report here that microarrays comprised of several thousand peptoids (oligo-N-substituted glycines) are useful tools for the identification of proteins via a "fingerprinting" approach. By using maltose-binding protein, glutathione S-transferase, and ubiquitin, a specific and highly reproducible pattern of binding was observed when fluorescently labeled protein was hybridized to the array. A similar pattern was obtained when binding of an unlabeled protein to the array was visualized by secondary hybridization of a labeled antibody against that protein, showing that native proteins can be identified without the requirement for prior chemical labeling. This work suggests that small-molecule microarrays might be used for more complex fingerprinting assays of potential diagnostic value.

Antibodies↗
PubMed2006

A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells.

In the subventricular zone of the adult mammalian forebrain, neural stem cells (NSCs) reside and proliferate to generate young neurons. We screened factors that promoted the proliferation of NSCs in vitro by a recently developed proteomics technique, the ProteinChip system. In this screen, we identified a soluble carbohydrate-binding protein, Galectin-1, as a candidate. We show herein that Galectin-1 is expressed in a subset of slowly dividing subventricular zone astrocytes, which includes the NSCs. Based on results from intraventricular infusion experiments and phenotypic analyses of knockout mice, we demonstrate that Galectin-1 is an endogenous factor that promotes the proliferation of NSCs in the adult brain.

Animals↗
PubMed2006

Abeta and tau form soluble complexes that may promote self aggregation of both into the insoluble forms observed in Alzheimer's disease.

To date, there is no reasonable explanation as to why plaques and tangles simultaneously accumulate in Alzheimer's disease (AD). We demonstrate here by Western blotting and ELISA that a stable complex can form between tau and amyloid-beta protein (Abeta). This complex enhances tau phosphorylation by GSK3beta, but the phosphorylation then promotes dissociation of the complex. We have localized the sites of this interaction by using peptide membrane arrays. Abeta binds to multiple tau peptides, especially those in exons 7 and 9. This binding is sharply reduced or abolished by phosphorylation of specific serine and threonine residues. Conversely, tau binds to multiple Abeta peptides in the mid to C-terminal regions of Abeta. This binding is also significantly decreased by GSK3beta phosphorylation of tau. We used surface plasmon resonance to determine the binding affinity of Abeta for tau and found it to be in the low nanomolar range and almost 1,000-fold higher than tau for itself. In soluble extracts from AD and control brain tissue, we detected Abeta bound to tau in ELISAs. We also found by double immunostaining of AD brain tissue that phosphorylated tau and Abeta form separate insoluble complexes within the same neurons and their processes. We hypothesize that in AD, an initial step in the pathogenesis may be the intracellular binding of soluble Abeta to soluble nonphosphorylated tau, thus promoting tau phosphorylation and Abeta nucleation. Blocking the sites where Abeta initially binds to tau might arrest the simultaneous formation of plaques and tangles in AD.

Aged↗
PubMed2004

FGF-2 induced by interleukin-1 beta through the action of phosphatidylinositol 3-kinase mediates endothelial mesenchymal transformation in corneal endothelial cells.

Our previous work demonstrated that both polymorphonuclear leukocytes (PMNs) and protein fractions released from PMNs induced de novo synthesis of fibroblast growth factor 2 (FGF-2), which in turn becomes the direct mediator of endothelial mesenchymal transformation observed in corneal endothelial cells (CECs). To identify the protein factor, we used ProteinChip Array technology. Protein fractions obtained from the conditioned medium released by PMNs were resolved by two-dimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 17 kDa, were sequentially subjected to in-gel trypsin digestion and mass spectrometry. The 17-kDa peptide band was identified as interleukin-1 beta (IL-1 beta). Biological activities of IL-1 beta were further determined; IL-1 beta altered the shape of CECs from polygonal to fibroblastic morphologies in a time- and dose-dependent manner, whereas neutralizing IL-1 beta antibody, neutralizing antibody to FGF-2, and LY294002 blocked the action of IL-1 beta. IL-1 beta greatly increased the levels of FGF-2 mRNA in a time- and dose-dependent manner; IL-1 beta stimulated expression of all isoforms of FGF-2. IL-1 beta initially induced nuclear accumulation of FGF-2 and facilitated translocation of FGF-2 to plasma membrane and extracellular matrix. IL-1 beta activated phosphatidylinositol (PI) 3-kinase, the enzyme activity of which was greatly stimulated after a 5-min exposure to IL-1 beta. This early and rapid activation of PI 3-kinase greatly enhanced FGF-2 production in CECs; pretreatment with LY294002 hampered the induction activity of IL-1 beta. These observations suggest that IL-1 beta takes part in endothelial to mesenchymal transformation of CECs through its inductive potential on FGF-2 via the action of PI 3-kinase.

Actins↗
PubMed2004

Pinpoint mapping of recognition residues on the cohesin surface by progressive homologue swapping.

The high affinity cohesin-dockerin interaction dictates the suprastructural assembly of the multienzyme cellulosome complex. The connection between affinity and species specificity was studied by exploring the recognition properties of two structurally related cohesin species of divergent specificity. The cohesins were examined by progressive rounds of swapping, in which corresponding homologous stretches were interchanged. The specificity of binding of the resultant chimeric cohesins was determined by enzyme-linked affinity assay and complementary protein microarray. In succeeding rounds, swapped segments were systematically contracted, according to the binding behavior of previously generated chimeras. In the fourth and final round we discerned three residues, reputedly involved in interspecies binding specificity. By replacing only these three residues, we were able to convert the specificity of the resultant mutated cohesin, which bound preferentially to the rival dockerin with approximately 20% capacity of the wild-type interaction. These residues represent but 3 of the 16 contact residues that participate in the cohesin-dockerin interaction. This approach allowed us to differentiate, in a structure-independent fashion, between residues critical for interspecies recognition and binding residues per se.

Amino Acid Sequence↗
PubMed2004

Systematic peptide array-based delineation of the differential beta-catenin interaction with Tcf4, E-cadherin, and adenomatous polyposis coli.

Nuclear accumulation of the complex between beta-catenin and proteins of the T-cell factor (Tcf) family is a hallmark of many cancers. Targeting this interaction for drug development is complicated by the fact that E-cadherin and adenomatous polyposis coli (APC) bind to overlapping sites on beta-catenin. Inhibiting their interactions might actually promote tumor growth. To identify selective beta-catenin binding hot spots of Tcf4, E-cadherin, and APC, array technology with peptides of up to 53 amino acids length was used. Interactions were monitored by a quantitative fluorescent readout, which was shown to represent a monitor of true equilibrium binding constants. We identified minimal binding motifs in the beta-catenin ligands and showed that most of the 15-mer and 20-mer repeats of APC did not interact, at least when non-phosphorylated, and defined a consensus binding motif also present in APC. We confirmed previously found hot spots and identified new ones. The method allowed us to locate a hydrophobic pocket that was relevant for the Tcf, but not the E-cadherin interaction, and would thus constitute an ideal drug target site.

Animals↗