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Phage display antibody-based proteomic device using resonance-enhanced detection.

The combination of phage display antibody arrays with a novel nanotransducer technique based on resonant nanoparticles in a nanosandwiched film enables the sensitive parallel screening of proteins. Using the resonance of nanoparticles with their induced mirror dipoles in a thin-film structure, limitations of fluorophores, such as unspecific background and nonvisibility to the eye, can be overcome, thereby leading to an optical signal significantly more sensitive than that of standard colloid techniques. The signal can be both directly observed as a color change of a microdot at the sensor surface and tuned throughout the visible range of the spectrum. Here we report the application of an optical chip using scFv-antibody-antigen interactions. Artificial scFv-antibodies against a variety of proteins, including yeast enzymes and bovine serum albumin (as a standard), were constructed via Phage Display. These scFv-antibodies were then coated onto metal nanoclusters and bound to their antigens that were arrayed as nanodroplets at the resonance layer of the chip. ScFv-Antibody-antigen interaction resulted in a visible array of microdots. Using resonance-enhanced absorption, the absorption signal of the spots was amplified by one to two orders of magnitude (compared to colloid-based techniques). For quantitative analysis, either an 8-micron scanner or a CCD camera (resolution 4 microns) was employed to gain direct-reflection spectra rather than unspecific scatter data (prone to dust and unspecific interaction). Our results demonstrate that this device enables high-throughput proteomics to overcome some limitations of fluorescence, enzyme labels, and colloid techniques.

Antibodies↗

PubMed2005

A pilot evaluation of the use of tissue microarrays for quantitation of target distribution in drug discovery pathology.

The use of tissue microarrays (TMAs) in the determination of novel target molecule distribution in organs is an expanding area of discovery pathology. This pilot study was carried out to assess the Chromavision automated cellular imaging system (ACIS) for quantitation of both mRNA and protein distribution in rat and dog TMAs. The targets chosen were a protein kinase, P-CIP2, for mRNA assessment and its downsteam target, peptidylglycine amidating monoxygenase (PAM), for immunohistochemistry (IHC). Oligonucleotide probes produced against P-CIP2, together with an antibody against PAM, were evaluated on rat and dog TMAs. A method for evaluation of target distribution using the ACIS was developed and involved a two-tier approach. Firstly, an initial scanning of the labelled slides identified which tissues expressed the target. Secondly, a more comprehensive analysis was made. This required operator interaction to select specific regions of interest within selected tissue cores and exclude any background labelling from the final assessment. This exacted the level of expression of P-CIP2 or PAM in different cellular populations in tissue cores. A comparative semi-quantitative analysis of the same arrays was concomitantly made by the pathologist in order to assess the relative benefits of a potentially time-consuming detailed morphological evaluation. This involved the histological identification by the pathologist of specific cell populations expressing P-CIP2 or PAM. In this study, we demonstrate the power of an image analysing system to provide quantitative data on target distribution by in situ hybridisation and IHC on normal TMAs. This methodology, together with detailed histological analysis by a pathologist, forms a guideline for future target distribution evaluation within discovery pathology.

Animals↗

PubMed2004

Simultaneous monitoring of multiple kinase activities by SELDI-TOF mass spectrometry.

Cellular response to the external environment is often controlled by one or more protein kinases. We report a methodology for simultaneously monitoring multiple kinase activities across multiple signal-transduction pathways using ProteinChip Array technology. Based on the addition of specific peptide reporters, kinase activity is detected by the presence of a mass shift of 80 Da (or multiple thereof) corresponding to the addition of one or more phosphate groups. These phosphorylated peptide substrates are then enriched using an immobilized metal affinity capture (IMAC)-Ga array and detected directly by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). SELDI-TOF MS is sensitive, tagless (nonradioactive, nonfluorescent), can be easily multiplexed for the analysis of several different kinases in a single reaction mixture (limited only by the specificity of the kinase for its substrate peptides), and is directly scalable through the use of robotic sample processing. By multiplexing kinase assays, one can dramatically increase the amount of information obtained from rare or volume-limited samples. More important, results reflect closely the complex interrelationships between kinases and show high correlation with in vivo assays.

Enzyme Inhibitors↗

PubMed2006

A novel approach of protein immobilization for protein chips using an oligo-cysteine tag.

Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamond-like, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.

Cysteine↗

PubMed2003

Protein microarrays guide tolerizing DNA vaccine treatment of autoimmune encephalomyelitis.

The diversity of autoimmune responses poses a formidable challenge to the development of antigen-specific tolerizing therapy. We developed 'myelin proteome' microarrays to profile the evolution of autoantibody responses in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). Increased diversity of autoantibody responses in acute EAE predicted a more severe clinical course. Chronic EAE was associated with previously undescribed extensive intra- and intermolecular epitope spreading of autoreactive B-cell responses. Array analysis of autoantigens targeted in acute EAE was used to guide the choice of autoantigen cDNAs to be incorporated into expression plasmids so as to generate tolerizing vaccines. Tolerizing DNA vaccines encoding a greater number of array-determined myelin targets proved superior in treating established EAE and reduced epitope spreading of autoreactive B-cell responses. Proteomic monitoring of autoantibody responses provides a useful approach to monitor autoimmune disease and to develop and tailor disease- and patient-specific tolerizing DNA vaccines.

Animals↗

PubMed2006

Proteome analysis to study signal transduction of G protein-coupled receptors.

G protein-coupled receptors (GPCR) play an important role in drug development. Although many classical signal transduction pathways have been elucidated, more and more cross-talk to other cascades, e.g. MAP-kinase have been reported. In order to identify the overall function of receptor stimulation in a specific cell type or under certain conditions proteome analysis has been shown to be a very successful and powerful approach. Here, we will summarize the current state of the art of proteome analysis applied to GPCR.

Animals↗

PubMed2005

Microarrays for the functional analysis of the chemical-kinase interactome.

A central challenge in chemical biology is profiling the activity of a large number of chemical structures against hundreds of biological targets, such as kinases. Conventional 32P-incorporation or immunoassay of phosphorylated residues produces high-quality signals for monitoring kinase reactions but is difficult to use in high-throughput screening (HTS) because of cost and the need for well-plate washing. The authors report a method for densely archiving compounds in nanodroplets on peptide or protein substrate-coated microarrays for subsequent profiling by aerosol deposition of kinases. Each microarray contains over 6000 reaction centers (1.0 nL each) whose phosphorylation progress can be detected by immunofluorescence. For p60c-src, the microarray produced a signal-to-background ratio of 36.3 and Z' factor of 0.63 for HTS and accurate enzyme kinetic parameters (KmATP = 3.3 microM) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 microM adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coated microarrays was inhibited in the nanoliter reactions by GW5074 at the expected IC50 of 9 nM. Common kinase inhibitors were printed on microarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFRbeta, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC50 determinations, and selectivity profiling.

Biological Assay↗

PubMed2006

New p63 targets in keratinocytes identified by a genome-wide approach.

p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The DeltaNp63alpha isoform is thought to play a 'master' role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing DeltaNp63alpha; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.

Animals↗

PubMed2006

Protein chip discovery of secreted proteins regulated by the phosphatidylinositol 3-kinase pathway in ovarian cancer cell lines.

Ovarian cancer has the highest mortality among the gynecologic malignancies. The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated, leading to increased cell survival. This study aimed to identify secreted proteins regulated by the PI3K pathway in ovarian cancer cell lines. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry with cation-exchange protein-chips was used to analyze secreted proteins from five ovarian cancer cell lines (SKOV-3, PE01, OVCAR-3, OV167, and OV207). To activate the PI3K pathway, cells were treated with 50 ng/mL epidermal growth factor (EGF) with or without 10 micromol/L LY294002, a PI3K inhibitor. Proteins induced by EGF and inhibited by LY294002, in the m/z range 7,500 to 9,500, were purified chromatographically, identified by peptide mass fingerprinting and NH(2)-terminal sequencing, and confirmed by immunodepletion. Two immunologically related proteins, m/z approximately 8,385 and 8,922, were identified as truncated and intact forms, respectively, of interleukin 8, a chemokine previously shown to be elevated in serum of ovarian cancer patients. Another protein, m/z 7,866, was identified as CXC chemokine ligand 1 (CXCL1) or GRO-alpha, a chemokine associated with melanoma formation and some epithelial cancers. EGF-stimulated CXCL1 levels were variably decreased by mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase and p38 MAPK inhibition in the five cell lines, but only LY294002 fully reversed the EGF effect in all cell lines. Immunoreactive CXCL1 levels in 160 conditioned media were highly correlated with corresponding peak intensities at m/z 7,866 by mass spectrometry, indicating the quantitative nature of these analyses. We conclude that proteomic analysis of cell models of human disease may facilitate the discovery of pathway-dependent proteins.

Blotting, Western↗

PubMed2006

SELDI-TOF MS analysis of the Cardiac Troponin I forms present in plasma from patients with myocardial infarction.

The troponin (Tn) complex is composed of troponin T, troponin C and troponin I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64 patients with acute myocardial infarction (AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips arrays. Upfront immunoaffinity enrichment using anti-cTnI 19C7 mAb allowed us to detect cTnI and bis-phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8 ng/mL. Cardiac troponin C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis-phosphorylated cTnI were mostly under the free form. Besides, a 18 718 m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC.

Blotting, Western↗

PubMed2002

Induction of GADD45 and GADD153 in neuroblastoma cells by dopamine-induced toxicity.

Dopamine (DA) metabolism and oxidation produce both reactive oxygen species (ROS) and reactive quinones. These chemical species are implicated in dopamine neurotoxicity and neurodegeneration. In the present studies, human neuroblastoma (SK-N-SH) cells were exposed to toxic concentrations of dopamine (333 microM) in order to investigate molecular pathways involved in dopamine toxicity. cDNA hybridization array (microarray) technology demonstrated that GADD45 and GADD153 (growth arrest and DNA-damage inducible) gene expression was increased in dopamine-treated cells (333 microM for 18 h). Subsequent Northern and Western blot analysis confirmed these changes in GADD45 and GADD153 gene expression. The antioxidant, ascorbic acid, significantly reduced the increase in GADD45 gene expression but did not significantly reduce GADD153 gene expression. Currently, the precise function of the GADD gene products is not known. It is known, however, that these genes are upregulated in response to stress to allow cells time to repair macromolecular damage. In the present case, GADD gene expression (manifested as increased mRNA and protein levels) preceded dopamine-induced cytotoxicity. It appears that dopamine, through the formation of reactive oxygen species and quinones, may damage cellular macromolecules to the point of inducing GADD gene expression. Other genes that displayed changes, but that have not been subjected to post-hoc confirmation, include clusterin (increased), ubiquitin (increased), CD27 ligand (increased), CD27BP (increased), and rac-PK-beta (decreased).

Ascorbic Acid↗

PubMed2004

Profiling the immune responses of human patients treated with recombinant streptokinase after myocardial infarct.

The SPOT synthesis of peptide arrays on continuous cellulose membranes should be generally applicable in the analysis of sequential antibody binding sites using the enzyme-substrate or other standard detection protocols. The use of total serum is limited by the occurrence of high background levels. This may be overcome if affinity purified antibodies or sera with high antibody titers are used, which allows work at high dilutions and a consequent reduction of background level. Here we demonstrate the mapping of antigenic regions located on recombinant streptokinase SK-2 (Heberkinase) using cellulose-bound peptide scans and human total sera from patients treated with SK-2 (Heberkinase). Streptokinase (SK) is a 47 kDa protein produced by various strains of hemolytic streptococci and is a potent activator of the fibrinolytic enzyme system in humans. SK is in widespread clinical use to treat acute infarction because of its function as an activator of vascular fibrinolysis. Since streptococcal infections are common, normal individuals are immunized with SK and antibodies (Abs) to SK can be detected in most of them. This therapy generates significant T-cell responses to SK and the neutralizing capacity of the Abs rises significantly. Neutralizing Abs reduces the efficiency of thrombolytic therapy and may cause allergic reactions. The widespread use of SK in humans makes its antigenicity an important clinical problem. In this regard the study of the immunodominant regions of SK becomes an important aspect for the improvement of this thrombolytic agent.

Amino Acid Sequence↗

PubMed2006

Human body fluid proteome analysis.

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.

Acute-Phase Proteins↗

PubMed2004

Serum protein profiling by SELDI mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients.

The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 alpha and serum amyloid alpha-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.

Adult↗

PubMed2004

Use of surface-enhanced laser desorption/ionization -time of flight to explore bacterial proteomes.

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is a recent technology that allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. We compared three simple procedures for protein extraction from bacteria for compatibility with the ProteinChip Array; we also determined the amount of protein required for each analysis. The protocol for the SELDI analysis was evaluated by generating protein expression profiles of a Streptococcus pneumoniae strain grown in different conditions and those of different strains of the same species. The protocol also was successfully applied to a wide range of Gram positive and negative bacteria. The results of this study suggest the appropriateness of this technology for microorganism protein profiling as complementary or alternative to two-dimensional gel electrophoresis.

Bacterial Proteins↗

PubMed2003

Microarray analysis and description of SMR1 gene in rat penis in a post-radical prostatectomy model of erectile dysfunction.

PURPOSE: We focused on the post-radical prostatectomy model to advance the understanding of neurogenic erectile dysfunction. We attempted to identify previously undescribed molecular changes via gene discovery methods using GeneChip (Affymetrix, Santa Clara, California) microarray technology. MATERIALS AND METHODS: Five male adult 120-day-old Sprague-Dawley rats underwent bilateral cavernous nerve neurectomy. Five age matched controls were prepared simultaneously. The penises were harvested on postoperative day 2 and snap frozen in liquid nitrogen. RNA was prepared and pooled into cut and uncut groups. Synthesis of cRNA was performed according to the GeneChip technical manual. Microarray analysis was performed on a U34A Rat Array (Affymetrix). This array has approximately 8,800 gene probe sets, approximately 6,600 known genes and approximately 2,200 estimated sequence transcripts. RESULTS: Dramatic results were found during GeneChip microarray expression analysis. A total of 126 candidate genes were noted to be altered based on the magnitude of expression change using rigorous statistical criteria, including 47 that were down-regulated and 79 that were up-regulated. Among the many significant changes seen 1 dominant class of genes was the submandibular rat genes. Submandibular rat 1 (SMR1) was down-regulated 82.5 fold. Other genes in this family were down-regulated 226 and 90 times. This result was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. These assays verified decreases in SMR1 at multiple time points after surgery. CONCLUSIONS: Impressive and previously unrecognized genetic changes are being intensely investigated as they are being unmasked by GeneChip technology. We have identified and begun the investigation of 1 interesting family of genes, namely submandibular gland proteins. The role of SMR as a clinically relevant change in penile and/or urethral function following cavernous nerve injury is speculative.

Animals↗

PubMed2006

Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments.

Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni(2+)-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(His)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(His)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays.

Antibodies↗

PubMed2004

Automated quantitative analysis of HDM2 expression in malignant melanoma shows association with early-stage disease and improved outcome.

The incidence of cutaneous malignant melanoma continues to increase every year, and this disease remains the leading cause of skin cancer death in industrialized countries. Despite the aggressive nature of advanced melanoma, there are no standard biological assays in clinical usage that can predict metastasis. This may be due, in part, to the inadequacy of reproducible assessment of protein expression using traditional immunohistochemistry. We have previously described a novel method of quantitative assessment of protein expression (AQUA) with the continuity and accuracy of an ELISA assay but with maintenance of critical spatial information. Here, we modify this technology for the evaluation of protein expression in melanoma. Using a tissue microarray cohort of 405 melanoma lesions and 17 normal skin samples, we analyzed expression of HDM2, the human homologue of murine double minute 2 with automated quantitative analysis. We show that expression levels in the nucleus are significantly higher in primary melanomas than in metastatic lesions. Furthermore, high levels of expression are predictive of better outcome. This study demonstrates that quantitative assessment of protein expression is useful in melanoma to validate potential tissue biomarkers and suggests that human homologue of murine double minute 2 may be a valuable prognostic tool for management of malignant melanoma.

Biomarkers, Tumor↗