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PubMed2003

Protein patterning on silicon-based surface using background hydrophobic thin film.

A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOP). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 microg/ml of total protein concentration, the CYTOP hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin-biotin system.

Biosensing Techniques↗
PubMedDate not supplied

Detection of the p110 beta subunit of phosphatidylinositol 3-kinase complexed with neutral endopeptidase.

BACKGROUND: Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that inactivates a variety of neuropeptide substrates. In addition to catalytic activity, NEP can exert biological effects through protein-protein interactions. We previously reported that NEP directly associated with tyrosine-phosphorylated Lyn kinase, and with the p85 subunit of the phosphatidylinositol 3-kinase (PI3 kinase) resulting in an NEP-Lyn-PI3 kinase protein complex. MATERIALS AND METHODS: In this report, we investigated the association of NEP with cytoplasmic proteins using ProteinChip Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry, as well as immunoprecipitation and Western blottings. RESULTS: Using immunocapture on the ProteinChip surface, we identified a 122 kDa protein which associates with NEP derived from LNCaP cell lysates which had the identical molecular weight as the beta-subunit of p110 subunit of phosphatidylinositol 3-kinase. The identity of the p110 beta was confirmed by Western blot analysis of NEP and p110 beta immunoprecipitates using monoclonal antibodies specific for NEP and p110 beta. CONCLUSION: These data confirm the association of phosphatidylinositol 3-kinase (consisting of the p85 adaptor and p110 beta-subunit) with NEP. Furthermore, this work demonstrates the ability of mass spectrometry to identify proteins interacting with NEP and potentially other cell-surface peptidases.

Blotting, Western↗
PubMed2003

Defining interacting partners for drug discovery.

Over the past few years, several technologies have been developed to determine interacting partners of proteins. The techniques fall into two broad categories: direct and indirect. Experimental techniques have been developed to directly probe protein interactions by monitoring protein-binding events. These techniques include the two-hybrid approach, protein fragment complementation assays, co-purification techniques and protein chips. In addition to these methodologies, several approaches have also emerged over the past few years to deduce indirect couplings between proteins. These couplings do not necessarily imply that two proteins are bound within the cell; however, they do provide evidence that perturbing one protein is likely to significantly perturb the function of its partner. These couplings may be deduced by studying the evolution of protein pairs, estimating the degree of correlated transcription of two genes, searching for synthetic lethal pairs, or identifying the chromosomal binding sites of transcriptional regulators. In all cases, protein interactions and protein couplings are being used to advance drug discovery by providing detailed information on protein functions, and by suggesting novel targets that act within biochemical pathways implicated in disease.

Drug Design↗
PubMed2003

Structural study of GCDFP-15/gp17 in disease versus physiological conditions using a proteomic approach.

Gross cystic disease fluid protein (GCDFP-15), also known as prolactin-inducible protein (PIP), is a specific breast tumor marker. GCDFP-15/PIP is also identified as gp17 and/or seminal actin-binding protein (SABP) from seminal vesicles and as extraparotid glycoprotein (EP-GP) from salivary glands. It is an aspartyl proteinase able to specifically cleave fibronectin (FN), suggesting a possible involvement in mammary tumor progression and fertilization. Other functions were attributed to this protein(s) on the basis of its ability to interact with an array of molecules such as CD4, actin, and fibrinogen. We investigated the structure of the protein(s) under disease versus physiological conditions by RP-HPLC chromatography, ProteinChip technology, and QStar MS/MS mass spectrometry. The proteins behaved differently when examined by RP-HPLC chromatography and surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry, suggesting different conformations and/or tissue-specific posttranslational modifications of the proteins, although their primary structure was identical by MS/MS analysis. Both showed a single N-glycosylation site. A different N-linked glycosylation pattern was observed in pathological GCDFP-15/PIP as compared with physiological gp17/SABP protein by coupling enzymatic digestion and ProteinChip technology. Furthermore, taking advantage of ProteinChip technology, we analyzed the interaction of both proteins with CD4 and FN. We observed that the physiological form was mainly involved in the binding to CD4. Moreover, we defined the specific FN binding-domain of this protein. These data suggested that, depending on its conformational state, the protein could differently bind to its various binding molecules and change its function(s) in the microenviroments where it is expressed.

Apolipoproteins↗
PubMed2003

Multiplexed protein profiling on antibody-based microarrays by rolling circle amplification.

Multiplexed immunoassays on antibody-based protein microarrays are an attractive solution for analyzing biological responses in normal and diseased states. Recently, the feasibility and utility of these assays has been established as concerns about specificity and sensitivity are being overcome by careful quality control and amplification technologies such as rolling circle amplification (RCA). RCA-amplified protein chips can now profile up to 150 proteins in various substrates including serum, plasma, and supernatants with high sensitivity, broad dynamic range and good reproducibility. Diagnostic utility of RCA-amplified protein chips has been shown for multiplexed allergen testing. When allied with multivariate statistical analysis, RCA protein chips have the potential to identify multiplexed biomarker classifiers for disease diagnosis and drug response.

Amino Acid Sequence↗
PubMed2003

Going global: protein expression profiling using shotgun mass spectrometry.

Protein expression profiling, the science of monitoring global sets of proteins produced by any given cell type, tissue or organism, has been invigorated by the introduction of proteomic technologies capable of characterizing large numbers of proteins. This review summarizes recent advances in mass spectrometry-based techniques for high-throughput protein identification and quantitation that are fueling rapid growth in the field. Key publications applying state-of-the-art 'shotgun' methods for investigating the entire protein complement of whole organelles, cells and tissues are highlighted. An overview of current proteomic challenges, particularly in the area of data analysis, and the long-term prospects of protein profiling strategies in basic biomedical research, therapeutics development and clinical discovery is also provided.

Animals↗
PubMed2002

Preparative protein refolding.

The rapid provision of purified native protein underpins both structural biology and the development of new biopharmaceuticals. The dominance of Escherichia coli as a cellular biofactory depends on technology for solubilizing and refolding proteins that are expressed as insoluble inclusion bodies. Such technology must be scale invariant, easily automated, generic for a broad range of similar proteins and economical. Refolding methods relying on denaturant dilution and column-based approaches meet these criteria. Recent developments, particularly in column-based methods, promise to extend the range of proteins that can be refolded successfully. Developments in preparing denatured purified protein and in the analysis of protein refolding products promise to remove bottlenecks in the overall process. Combined, these developments promise to facilitate the rapid and automated determination of appropriate refolding conditions and to simplify scale-up.

Chromatography, Affinity↗
PubMed2002

Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry.

Mass spectrometry can be used to map the distribution of targeted compounds in tissue, providing important molecular information in many areas of biological research. Matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) is well suited for the analysis of tissue samples with a spatial resolution of about 30 microm for compounds in a mass range from 1000 to over 50 000 Da. Direct analysis of tissue sections requires spotting or coating of the tissue with a matrix compound typically sinapinic acid or other cinnamic acid analogs. A raster of this sample by the laser beam and subsequent mass analysis of the desorbed ions can record molecular intensities throughout the section. The overall process is illustrated by profiling and imaging of mouse epididymis sections where protein activity changes markedly throughout the section.

Animals↗
PubMed2003

Protein microarrays: new tools for pharmaceutical development.

Protein microarrays are a relatively new technology, which will dramatically impact the pharmaceutical industry. The critical need for more rapid identification of novel drug targets, and for obtaining high-quality information early in the target validation process is a major driver for the industry. High-throughput protein analytical techniques are critical for obtaining biological information beyond that which transcript analysis can provide, given that proteins are the "worker bees" in cells. The vast complexity of proteins when compared to DNA and RNA in terms of sheer number, and structural and biochemical diversity requires a higher degree of sophistication in both assay design and data analysis. High-throughput microarray technology platforms allow for simultaneous, multi-parametric analysis of complex protein mixtures. Protein microarrays have tremendous potential as a tool for the study of protein-protein, enzyme-substrate, and antibody-antigen interactions among others. They can also be used for biomarkers and drug target identification via comparative proteomic analysis of healthy and disease tissues. More recently, cellular microarrays that enable identification of cell-surface receptors and other cell-surface proteins allowing rapid screening of cell-specific, novel drug targets, are being developed. This review will focus on the technical issues and potential applications of protein microarrays in pharmaceutical discovery.

Drugs, Investigational↗
PubMed2002

From one well to 9000: using high-density streptavidin-coated membranes for kinase detection.

The study of kinases and their role in cellular regulation continues to expand as the human genome is sequenced and new kinases are identified as expression products of newly discovered genes. Reagents and assay systems that allow for sensitive, accurate, and high-throughput analysis of both purified kinases as well as crude extracts will enhance the characterization of these important cellular components and will speed the identification of appropriate therapeutic targets and the development of new and more effective treatments.

Autoradiography↗
PubMed2003

DNA: a programmable force sensor.

Direct quantification of biomolecular interaction by single-molecule force spectroscopy has evolved into a powerful tool for materials and life sciences. We introduce an approach in which the unbinding forces required to break intermolecular bonds are measured in a differential format by comparison with a known reference bond (here, a short DNA duplex). In addition to a marked increase in sensitivity and force resolution, which enabled us to resolve single-base pair mismatches, this concept allows for highly specific parallel assays. This option was exploited to overcome cross-reactions of antibodies in a protein biochip application.

Animals↗
PubMed2003

Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.

Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. In the past, analysis of global protein phosphorylation has been accomplished through radiolabelling of samples with inorganic (32P or [gamma-32)P] ATP. The approach is limited to specimens amenable to radiolabelling and poses certain safety and disposal problems. Alternatively, immunodetection with antibodies to the common phosphoamino acids may be employed, but the antibodies are relatively expensive and exhibit limited specificity and a certain degree of cross-reactivity. Pro-Q Diamond dye is a new fluorescent phosphosensor technology suitable for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins directly in isoelectric focusing gels, SDS-polyacrylamide gels and two-dimensional gels. Additionally, the technology is appropriate for the detection of phosphoproteins or phosphopeptides arrayed on protein chips or affixed to beads. Dye-stained proteins and peptides can be excited with a laser-based light source of 532 or 543 nm or with a xenon-arc lamp-based system equipped with appropriate band pass filters. Alternatively, ultraviolet light of about 302 nm may be employed, providing that sufficiently long exposure times are used to collect the fluorescence signal. Pro-Q Diamond dye emits maximally at approximately 580 nm. The fluorescence-based detection technology is easy to conduct, cost effective and allows rapid large-scale screening of protein and peptide phosphorylation in a variety of solid-phase assay formats.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Tagless extraction-retentate chromatography: a new global protein digestion strategy for monitoring differential protein expression.

A new global protein digestion and selective peptide extraction strategy for the purpose of monitoring differential protein expression, coined as tagless extraction-retentate chromatography, is introduced. Target protein populations are firstly digested under reduced and alkylated conditions, and resultant peptides selectively extracted via covalent attachment to methionine residues by bromoacetyl reactive groups tethered to the surface of glass beads packed in small reaction vessels. After conjugation, reactive beads are stringently washed to remove nonspecifically bound peptides and then later treated with beta-mercaptoethanol to release captured methionine peptides in their nascent state, without complicating affinity tags. Recovered methionine containing peptides are profiled using the surface-enhanced laser desorption/ionization (SELDI) retentate chromatography mass spectrometry (RCMS) method. Selected peptides are further studied employing ProteinChip tandem mass spectrometry (MS/MS) analysis to identify their parent proteins. This approach has been applied to an Escherichia coli lysate model system and has demonstrated facility in reducing global digest complexity, sensitivity to low protein expression levels, and significant quantitative capability. It is envisioned that tagless extraction-RCMS will evolve to be a valuable approach for both basic research and clinical proteomics endeavors.

Amino Acid Sequence↗
PubMed2003

Applications of one-bead one-compound combinatorial libraries and chemical microarrays in signal transduction research.

The "one-bead-one-compound" (OBOC) combinatorial library method synthesizes millions of random compounds such that each bead displays only one compound. Bead libraries are screened, and positive beads are isolated for structure analysis. Peptide substrates and inhibitors of protein kinases, and peptide ligands for cell surface receptors have been identified using this method. A novel encoding strategy for OBOC libraries has been developed to identify peptidomimetic and small-molecule ligands that specifically interact with cellular proteins. These ligands will be tested for their effects on cell signaling and used to construct chemical microarrays for further characterization of ligand-protein interactions.

Combinatorial Chemistry Techniques↗
PubMed2002

Proteomic approaches for the global analysis of proteins.

Improvements in technology that allow miniaturization and high-throughput analyses of thousand of genes and gene products have changed the focus and scope of research and development in both academia and industry. It is now possible to study entire proteomes with the goals of elucidating protein expression, subcellular localization, biochemical activities, and their regulation. Alterations in different cell types and conditions and in normal and disease states can be revealed. This wealth of information not only has facilitated our basic understanding of many biological processes but also has enormous potential for drug discovery and development.

Data Collection↗
PubMed2002

Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.

Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.

Protein Array Analysis↗
PubMed2003

Molecular evaluation using in silico protein interaction profiles.

MOTIVATION: To find a correlation between the activities and structures of molecules is one of the most important subjects for molecular evaluation study. Traditional quantitative structure-activity relationship (QSAR) methodologies represent those attempts using physicochemical descriptors. Creating a new molecular description factor based on the results of a computational docking study will add new dimensions to molecular evaluation. RESULTS: We propose a new molecular description factor analysis system called the Comparative Molecular Interaction Profile Analysis (CoMIPA) system in which the AutoDock program is used for docking evaluation of small molecule compound-protein complexes. Interaction energies are calculated, and the data sets obtained are called interaction profiles (IPFs). Using the IPF as a scoring indicator, the system could be a powerful tool to cluster the interacting properties between small molecules and bio macromolecules such as ligand-receptor bindings. Further development of the system will enable us to predict the adverse effects of a drug candidate.

Algorithms↗
PubMed2003

Development of protein microarray technology to monitor biomarkers of rheumatoid arthritis disease.

Most biological processes are mediated by complex networks of molecular interactions involving proteins. The analysis of protein expression in biological samples is especially important in the identification and monitoring of biomarkers for disease progression and therapeutic endpoints. In this paper, the development of a protein microarray format for multiplexed quantitative analysis of several potential markers for rheumatoid arthritis (RA) is described. Development of a high-performance protein microarray system depends on several key parameters such as surface chemistry, capture agents, immobilization technology, and methods used for signal detection and quantification. Several technical possibilities were investigated and compared: poly-L-lysine versus self-assembled monolayer of octadecyl phosphoric acid ester for surface chemistries; noncontact piezoelectric versus contact printing technology for antibody deposition; CCD camera capture versus fluorescent scanning for image detection; and the concentration of coating antibody. On the basis of reproducibility, signal-to-noise ratio, and sensitivity we have selected self-assembled monolayer, noncontact piezoelectric printer, and high-read-out fluorescence scanning for our microarray format. This format was used to perform multiplexed quantitative analysis of several potential markers of disease progression of rheumatoid arthritis: IL-1beta, IL-6, IL-8, MCP-1, and SAA. Some assays, such as MCP-1, provided a working range that covered physiologically relevant concentrations. Other assays, such as IL-6 and SAA, lacked sensitivity or were too sensitive for measuring biological concentrations, respectively. The results described demonstrate the applicability of protein microarrays to monitor RA markers; however, sandwich assay methodologies need to be further optimized to measure the appropriate biological ranges of these markers on one chip.

Arthritis, Rheumatoid↗