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At least 523 records · Page 29Linked to original sources
PubMed2004

Capture and release of proteins on the nanoscale by stimuli-responsive elastin-like polypeptide "switches".

This article describes the fabrication and characterization of stimulus-responsive elastin-like polypeptide (ELP) nanostructures grafted onto omega-substituted thiolates that were patterned onto gold surfaces by dip-pen nanolithography (DPN). In response to external stimuli such as changes in temperature or ionic strength, ELPs undergo a switchable and reversible, hydrophilic-hydrophobic phase transition at a lower critical solution temperature (LCST). We exploited this phase transition behavior to reversibly immobilize a thioredoxin-ELP (Trx-ELP) fusion protein onto the ELP nanopattern above the LCST. Subsequent binding of an anti-thioredoxin monoclonal antibody (anti-Trx) to the surface-captured thioredoxin showed the presentation of the immobilized protein in a sterically accessible orientation in the nanoarray. We also showed that the resulting Trx-ELP/anti-Trx complex formed above the LCST could be reversibly dissociated below the LCST. These results demonstrate the intriguing potential of ELP nanostructures as generic, reversible, biomolecular switches for on-chip capture and release of a small number (order 100-200) of protein molecules in integrated, nanoscale bioanalytical devices. We also investigated the molecular mechanism underlying this switch by measuring the height changes that accompany the binding and desorption steps and by adhesion force spectroscopy using atomic force microscopy.

Elastin↗
PubMed2006

Transport at the air/water interface is the reason for rings in protein microarrays.

Fabricating a protein microarray involves the deposition of nanoliter droplets of solutions on a solid surface. Instead of uniform spots, one often observes ring-like structures that add to the difficulty in quantification. We show that the accumulation of proteins at the air/water interface of the nanodroplet is the reason. Transformation to a uniform spot can be achieved via the addition of competitive surfactants or the control of surface reaction kinetics.

Air↗
PubMed2006

Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays.

One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.

Amino Acid Sequence↗
PubMed2006

Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids.

A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogues bearing omega-azide or omega-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click"-immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 degrees C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.

Amino Acids↗
PubMed2006

Circumventing the problems caused by protein diversity in microarrays: implications for protein interaction networks.

Protein microarrays provide a well-controlled, high-throughput way to uncover protein-protein interactions. One problem with this and other standardized assays, however, is that proteins vary considerably with respect to their physical properties. If a simple threshold-based approach is used to define protein-protein interactions, the resulting binary networks can be strongly biased. Here, we investigate the extent to which even closely related protein interaction domains vary when printed as microarrays. We find that, when a collection of well behaved, monomeric Src homology 2 (SH2) domains are printed at the same concentration, they vary by up to 50-fold with respect to the resulting surface density of active protein. When a threshold-based binding assay is performed on these domains using fluorescently labeled phosphopeptides, a misleading picture of the underlying biophysical interactions emerges. This problem can be circumvented, however, by obtaining saturation binding curves for each protein-peptide interaction. Importantly, the equilibrium dissociation constants obtained from these curves are independent of the surface density of active protein. We submit that an increased emphasis should be placed on obtaining quantitative information from protein microarrays and that this should serve as a more general goal in all efforts to define large-scale protein interaction networks.

Biosensing Techniques↗
PubMed2004

Physiological difference between free and triglyceride-type conjugated linoleic acid on the immune function of C57BL/6N mice.

Previous studies have shown the physiological significance of dietary conjugated linoleic acid (CLA) in various experimental animals and in human beings. One of the important problems to better elucidate is the difference between triglyceride (TG) and free (FFA) dietary CLA. Here, using splenocytes, this study assesses how TG- and FFA-CLA modulate immunoglobulin and various cytokine productions. In this study, C57BL/6N mice were fed an experimental diet containing 0% CLA, 0.1 or 1% FFA-CLA, or 0.1 or 1% TG-CLA for 3 weeks. The production of immunoglobulin tended to be up-regulated by 1% FFA-CLA. As a result of protein array analysis using the supernatant from splenocytes cultured with no CLA, 1% FFA-CLA, and TG-CLA, some cytokine production was shown to be remarkably regulated by dietary FFA- and TG-CLA. A total of 32 cytokines were examined, and 11-14 produced cytokines that were 2-fold up-regulated as compared with control for FFA- or TG-CLA, respectively. Especially, the production of IL-9 and MCP-5 and other cytokines was remarkably up-regulated by both FFA- and TG-CLA. In addition, seven cytokines were 2-fold down-regulated by TG-CLA. These data show that there is a slight but significant difference between the functionalities of FFA- and TG-CLA.

Animals↗
PubMed2004

Dendrimer-activated surfaces for high density and high activity protein chip applications.

Highly functional Si and glass surfaces for protein immobilization have been prepared by a facile activation of native surface silanol groups. Poly(propyleneimine) dendrimers of generations 1-5 were immobilized onto the surface using a facile room-temperature coupling procedure that involved activation of native silanol groups of glass using 1,1'-carbonyldiimidazole under anhydrous conditions. The dendrimer-coated surfaces were used to immobilize proteins and were characterized with respect to surface loading and activity. A number of different chemical, physical, and biochemical techniques including contact angle measurement, ellipsometry, and fluorescence microscopy were used to characterize the resulting surfaces. Increasing the dendrimer generation past G-3 led to increased surface amine content, immobilized protein concentration, and the activity of immobilized alkaline phosphatase (used as a test system). Very high activity of the immobilized proteins in the case of higher generation (G-4 and G-5) dendrimers led us to conclude that such an approach has true potential for creating highly functional surfaces for protein chip applications.

Alkaline Phosphatase↗
PubMed2006

Surface energy modified chips for detection of conformational states and enzymatic activity in biomolecules.

A novel patterning method for anchoring biomolecules and noncovalent assembled conjugated polyelectrolyte (CPE)/biomolecule complexes to a chip surface is presented. The surface energy of a hydrophilic substrate is modified using an elastomeric poly(dimethylsiloxane) (PDMS) stamp, containing a relief pattern. Modification takes place on the parts where the PDMS stamp is in conformal contact with the substrate and leaves low molecular weight PDMS residues on the surface resulting in a hydrophobic modification, and then biomolecules and CPE/biomolecule complexes are then adsorbed in a specific pattern. The method constitutes a discrimination system for different conformations in biomolecules using CPEs as reporters and the PDMS modified substrates as the discriminator. Detection of different conformations in two biomacromolecules, a synthetic peptide (JR2E) and a protein (calmodulin), reported by the CPE and resolved by fluorescence was demonstrated. Also, excellent enzyme activity in patterned CPE/horseradish peroxidase (HRP) enzyme was shown, demonstrating that this method can be used to pattern biomolecules with their activity retained. The method presented could be useful in various biochip applications, such as analyzing proteins and peptides in large-scale production, in making metabolic chips, and for making multi-microarrays.

Calmodulin↗
PubMed2006

High-capacity binding of proteins by poly(acrylic acid) brushes and their derivatives.

Polymeric coatings with high protein-binding capacities are important for increasing the output of affinity-based protein purification and decreasing the detection limits of antibody microarrays. This report describes the use of thick poly(acrylic acid) (PAA) brushes to immobilize as much as 80 monolayers of protein. The brushes were prepared using a recently developed procedure that allows polymerization of 100-nm-thick poly(tert-butyl acrylate) films from a surface in just 5 min along with hydrolysis of these films to PAA in 15 min. Covalent binding of bovine serum albumin (BSA) to PAA brushes that were activated using standard coupling agents, however, resulted in immobilization of less than two monolayers of BSA because of competitive hydrolysis of the esters in the activated film. In contrast, derivatization of PAA with nitrilotriacetate (NTA)-Cu2+ complexes yielded films capable of binding many monolayers of protein via metal-ion affinity interactions. For example, derivatization of 55-nm-thick PAA films with NTA-Cu2+ allowed immobilization of about 15 monolayers (5.8 microg/cm2 or 58 nm) of BSA. The binding capacity was even higher for myoglobin (7.7 microg/cm2) and anti-IgG (9.6 microg/cm2). Remarkably, electrostatic adsorption of lysozyme in 55-nm-thick, underivatized PAA resulted in as much as 80 monolayers (16.2 microg/cm2 or 162 nm) of adsorbed protein. Polymer synthesis, derivatization, and swelling, as well as BSA immobilization kinetics and thermodynamics were characterized using reflectance FT-IR spectroscopy, ellipsometry, and protein assays.

Acrylic Resins↗
PubMedDate not supplied

Antibody detection in human serum using a versatile protein chip platform constructed by applying nanoscale self-assembled architectures on gold.

We report a novel high-throughput (HTP) protein chip platform, constructed on gold using self-assembly techniques, for conducting high quality antigen-antibody interactions. Biotinylated monolayers were used to immobilize a streptavidin surface with high packing density. This biocompatible platform was then used for detection of serum IgM antibodies. Serum samples of patients suspected to suffer from Lyme borreliosis were used to validate the protein chip platform using biotinylated peptide AAOspC8 molecules as the test probes. Various experimental parameters such as the effect of concentration of probes, targets, temperature of incubation, and their effect on the resulting signal-to-noise ratio are described in detail. Highly specific protein interaction data with a high signal-to-noise ratio were obtained with serum sample solutions as low as 1 microL/spot (1/10 diluted).

Antibodies, Bacterial↗
PubMedDate not supplied

High-throughput, fluorescence-based screening for soluble protein expression.

Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

Boron Compounds↗
PubMedDate not supplied

Integrated protein microchip assay with dual fluorescent- and MALDI read-out.

A pore chip protein array (PCPA) concept based on a dual readout configuration, fluorescence imaging, and MALDI-TOF MS has been developed. Highly packed, (>4000 spots/cm2), antibody arrays were dispensed on the porous chip by using a piezo-electric microdispenser. Sandwich assay was made after blocking by addition of a secondary antibody either IgG-FITC-labeled or anti-Ang II. The antigen in the first system was a large protein (IgG), and in the other system, a FITC marked peptide Angiotensin II (Ang II) was used. Ang II antibodies showed specificity for Ang II, while the Ang I antibodies showed binding properties for Ang I, II, and Renin. Fluorescence and MALDI TOF MS read-out was made for IgG and Ang II. A major advantage of the dual read-out PCPA approach is that both affinity binding and mass identity are derived. Detection limits for Ang II on the chip is as low as 500 zmol (Ang II).

Angiotensin I↗
PubMedDate not supplied

Differential protein expression profiles of gastric epithelial cells following Helicobacter pylori infection using ProteinChips.

Helicobacter pylori infects approximately half of the world's population and the bacterium is associated with gastric cancer and peptic and duodenal ulcers. In this study, Surface Enhanced Laser Desorption /Ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to identify the biomarkers from H. pylori infected gastric epithelial cells (GEC) to understand key mechanisms associated with pathogenesis. Using different chip surfaces, differential protein expression profile of GEC was obtained and several upregulated or downregulated biomarkers were detected on GEC, following H. pylori infection. Four different H. pylori infected GECs were compared based on their expression of MHC class II, a receptor reported to trigger apoptosis. One biomarker was identified in H. pylori infected GEC as Annexin A2 (Annexin II) from the flow through of the anion-exchange resin. The increased expression of Annexin II in GEC following H. pylori infection was further confirmed by Western Blot analyses and indicates its involvement in H. pylori pathogenesis.

Anion Exchange Resins↗
PubMedDate not supplied

One-pot glyco-affinity precipitation purification for enhanced proteomics: the flexible alignment of solution-phase capture/release and solid-phase separation.

A one-pot affinity precipitation purification of carbohydrate-binding protein was demonstrated by designing thermally responsive glyco-polypeptide polymers, which were synthesized by selective coupling of pendant carbohydrate groups to a recombinant elastin-like triblock protein copolymer (ELP). The thermally driven inverse transition temperature of the ELP-based triblock polymer is maintained upon incorporation of carbohydrate ligands, which was confirmed by differential scanning calorimetry and (1)H NMR spectroscopy experiments. As a test system, lactose derivatized ELP was used to selectively purify a galactose-specific binding lectin through simple temperature-triggered precipitation in a high level of efficiency. Potential opportunities might be provided for enhanced proteomic, cell isolation as well as pathogen detection applications.

Amino Acid Sequence↗
PubMed2006

Beyond linker histones and high mobility group proteins: global profiling of perchloric acid soluble proteins.

Extraction with HClO(4) provides an easy method for efficient enrichment of both histone H1 and HMG proteins from a variety of tissues. Usually, the histone and the HMG proteins are the most abundant components of the extracts, however, other proteins have frequently been observed but only seldom studied in more detail. Here we describe a study aimed at global characterization of HClO(4) extractable proteins from breast cancer cell lines. We report identification of 150 unique proteins by liquid chromatography tandem mass spectrometry including almost all major histone H1 variants and canonical members of the HMG protein families. In the extracts, diverse proteins with HMG-like amino acid composition were identified and their post-translational modifications were mapped. Importantly, those include multiple proteins known or supposed to be related to cell proliferation and cancer. Since purification of these proteins as well as low abundant variants of histone and HMG proteins is difficult due to their metabolic instability, characterization of these proteins from crude extracts can facilitate studies aimed at better understanding of their function.

Amino Acid Sequence↗
PubMed2006

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films.

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.

Animals↗
PubMed2004

Validation of a tissue microarray to study differential protein expression in inflammatory and non-inflammatory breast cancer.

AIMS: Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with poor prognosis. The mechanisms responsible for the aggressive clinical evolution are incompletely understood. We constructed a tissue microarray (TMA) and validated its use in translational IBC research. Differential expression of proteins that might play a role in causing the IBC phenotype was studied. METHODS AND RESULTS: A TMA containing 34 IBC and 41 non-stage matched non-IBC tumours was constructed. Five core biopsies were taken for each IBC and three cores for each non-IBC tumour. The TMA was validated using three approaches: (1) the excellent concordance between immunohistochemical results of the initial pathological examination and the results obtained with the TMA for ER, PR and HER2/neu (kappa > 0.74); (2) the known differential expression between IBC and non-IBC for four bio-markers in IBC (ER, PR, p53 and HER2/neu) was confirmed ( p < 0.01); (3) the HER2/neu status using three different antibodies (CB11, TAB250 and HercepTest) was highly concordant (kappa > 0.75). Furthermore, the overexpression of E-Cadherin and RhoC GTPase in IBC ( p < 0.05) was confirmed. We did not find a differential expression pattern for carbonic anhydrase IX (CA IX) and EGFR. CONCLUSIONS: Using different approaches, we have validated the use of our TMA for studying differential protein expression in IBC and non-IBC. We confirm the overexpression of E-Cadherin and RhoC GTPase in IBC. The lack of differential expression for CA IX and EGFR might suggest the pathways are equally utilised in both types of breast cancer.

Adenocarcinoma↗