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The massive throughput offered by array-based technologies can only be realized with the development of equally powerful strategies that offer reproducible consistency. The competence of arrays and efficacy of screening come under scrutiny, with most existing immobilization schemes that do not site-specifically ligate peptides on the arrays. Thus,...
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... Die Epoxy-funktionalisierten Crn Glasträger wurden, laut Hersteller, ebenfalls unter den experimentellen Bedingungen gedruckt, die für Aldehyd-funktionalisierte Glasträger (Gnx, Eri und Grn) optimiert wurden. Unter diesen Bedingungen verläuft der Immobilisierungsprozess auf Crn Glasträgern weniger chemoselektiv [89,153] und führt folglich zu einer geringeren Reinheit oder unregelmäßigen Dichte der bedruckten Peptide, was eine höhere Variabilität der Signalintensität zur Folge hat. ...
Die humorale Immunantwort eines Organismus auf ein Pathogen äußert sich in einer Veränderung des Antikörperrepertoires. Eine quantitative Untersuchung dieses Prozesses erfordert aufgrund der enormen Komplexität des Immunsystems, die Verwendung von Hochdurchsatztechniken, wie Peptid-Mikroarrays. Bisher gibt es nur wenige Studien, die die Verlässlichkeit von Mikroarray-Bindungsmessungen untersuchen. In dieser Arbeit werden Bewertungskriterien für die Qualität von Antikörper-Peptid-Bindungsstudien unter Verwendung der Peptid-Mikroarraytechnologie herausgearbeitet, mit dem Ziel, diese Hochdurchsatzmethode für qualitative und quantitative Antikörper-Peptid-Bindungsmessungen zu optimieren. Anhand eines Modellsystems, das aus dem monoklonalen anti-p24 (HIV-1) Antikörper CB4-1 und 26 verschiedenen Peptiden, die mit unterschiedlicher Affinität an CB4-1 binden, besteht, werden systematisch die Bindungsdissoziationskonstanten der jeweiligen Antikörper-Peptid-Komplexe mit den durch Peptid-Mikroarray-Bindungsmessungen erhaltenen Signalintensitäten verglichen. Darüber hinaus wird in dieser Arbeit die Messung von Serumantikörperbindungsprofilen gegenüber Zufallspeptidbibliotheken als Methode der serologischen Diagnostik verwendet. Anhand dreier Beispieldatensätze wird die serologische Diagnose von Infektionskrankheiten, Autoimmunkrankheiten und von Krebs mittels Zufallspeptid-Mikroarrays demonstriert. Mithilfe von Merkmalsselektion werden Peptide selektiert, die besonders geeignet sind, um zwischen gesunden und kranken Individuen zu unterscheiden. Besondere Bedeutung wird der Untersuchung der Robustheit der Methode gegenüber schwankenden experimentellen Bedingungen beigemessen. Die vorliegende Arbeit gibt Aufschluss über vorhandene Probleme der Mikroarray-Technologie, stellt Lösungsansätze vor und arbeitet bedeutende Weiterentwicklungen auf dem Weg hin zu einer minimal-invasiven serologischen Diagnostik heraus, die kein a priori Wissen über Antigene voraussetzt.
... More recently, protein-and peptide-based microarrays have also been developed (7)(8)(9)(10)(11). In comparison to SPOT technology, a much higher density of spots can be attained using a protein array, making it possible to simultaneously screen tens of thousands of kinase substrates on the solid surface. ...
... The customized microarray robot was also used to print arrays for the biotinylated peptides (Operon) in phosphate-buffered saline (pH 7.4). After printing, the arrays were incubated at RT for 4 h in a humidified chamber, washed, and treated with the blocking solution according to a previously reported method (11). ...
... The performance of different attachment chemistry (i.e., most widely used in streptavidin-biotin interaction) was also assessed by testing the detection sensitivity of phosphopeptide, as shown in Figure 5A and B. To this end, we immobilized a total of four different biotinylated peptides (same sequences as those of Peptides 1 and 2, and Peptides 4 and 5 in Table 1) at different concentrations onto streptavidin-coated glass slides and then probed with corresponding Cy5-labeled antiphosphoamino acid antibody (11). In our hands, however, we could not detect any signals for the phosphorylated Peptide 1 on the streptavidin slide using interaction and scanning conditions (laser power 100%, PMT 50%) identical to those used in our methodology (data not shown), probably due to the fact that the streptavidin-coated slide has limited peptide binding capacity and/or due to the steric interference of the surface on interaction with the antibody where the phosphorylated tyrosine residue in Peptide 1 is located adjacent to the slide surface (Table 1) (25). ...
The present study reported proof-of-principle for a kinase assay approach that can detect specific peptide phosphorylation events. The method involves attachment of peptides onto commercial aminosilane and polycarbodiimide-coated glass slides, using a newly developed DNattach linker system that consists of a poly(dT) tail (Nisshinbo Industries Inc.), followed by a detection step using fluorescently labeled antiphosphoamino acid antibodies. The linker-modified peptides are efficiently synthesized by Michael addition between maleimido-modified peptides and thiol-containing DNattach. Specific covalent immobilization of the modified peptides onto aminosilane and poly carbodiimide-coated slides is then achieved by short exposure to UV-light. Highly selective and quantitative recognition by standard antiphosphoamino acid antibodies (antiphosphotyrosine and anti-phosphoGFAP) and kinases (c-Src and PKA) to the corresponding modified peptides on the microarray spots is demonstrated. Furthermore, we found that this immobilization method provides greater signal-to-noise ratio and better discrimination ability of phosphorylated amino acids than does the conventional immobilization technique. The phosphorylation pattern of target sequences, detected using fluorescently labeled antiphosphoamino acid antibodies, revealed that the linker system preference of the kinase is determined by its activity profile.
... Despite the difficulties in handling small molecule arrays, microarraying peptides has demonstrated to be a very promising strategy for epitope mapping (Chiari et al., 2005), detection of pathogen infections (Duburcq et al., 2004) or signalling-dependent changes of molecular interactions (Stoevesandt et al., 2005). Enzymeprofiling arrays to analyze enzymatic activities, especially phosphorylation catalyzed by protein kinases, has been frequently reported (Uttamchandani et al., 2004). Peptide arrays have also been used to identify ligands that are active in cell adhesion (Falsey et al., 2001) and, in another interesting application aimed to develop peptidic inhibitors of b-lactamases, using a phage display library (Huang et al., 2003). ...
Microarrays of proteins and peptides make it possible the screening of thousands of binding events in a parallel and high throughput fashion; therefore they are emerging as a powerful tool for proteomics and clinical assays. The complex nature of Proteome, the wide dynamic range of protein concentration in real samples and the critical role of immobilized protein orientation must be taken into account to maximize the utility of protein microarrays. Immobilization strategy and designing of an ideal local chemical environment on the solid surface are both essential for the success of a protein microarray experiment. This review article will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications.
... Généralement, l'immobilisation chimie-sélective utilise des aldéhydes et des glyoxylyl comme groupements fonctionnels de la surface qui interagissent avec des groupements amino-oxy-acétyl des séquences peptidiques modifiées (Reimer et al., 2002;Rychlewski et al., 2004). Il est possible aussi de fixer directement les peptides par l'intermédiaire d'un résidu cystéine Lesaicherre et al., 2002b;Uttamchandani et al., 2003;Uttamchandani et al., 2004). Des liaisons type Diels-Alder entre des groupements de benzoquinone présents sur le support et des peptides modifiés avec une cyclopentadiène permettent une fixation covalente. ...
... Les résultats de mise au point des puces à peptides obtenues permettent de valider pour la première fois la chimie du pyrrole avec des sérums pour une analyse par imagerie SPR. Rappelons que pour des analyses sur des échantillons biologiques, un grand nombre de méthodes utilisent l'ELISA pour détecter les interactions peptides-protéines : celle-ci sont révélées par l'intermédiaire de réactions Ag-Ac et/ou avidine-biotine, couplé à un marquage fluorescent (Shreffler et al., 2004;Uttamchandani et al., 2004). Ce type de détection, coûteuse en temps et matériel, ne permet pas une analyse directe ni une mesure en temps réel avec une détermination des constantes d'affinité. ...
... –15-mer phosphotyrosine-containing peptides derived from human insulin receptor; degenerate SPOT library of general structure AABX 1 ZX 2 BAAA (Z = phosphotyrosine, B = mixture of 20 natural amino acids, X = defined amino acids), progressive alanine substitution of MTRDIYETDYZRKGG (Z = phosphotyrosine), alanine scan of TRDIYETDYZRKGGKGL, substitutional analysis for X in MTRDIYETDXZRKGG (Z = phosphotyrosine) c-Src and g 32 P-ATP or antiphosphotyrosine antibody panel of known kinase substrates DIPP [31] c-Src and g 32 P-ATP c-Src substrate DIPP [63] native chemical ligation, introduced by Dawson et al. [27] is well suited for effective attachment of kinase substrate peptides containing an N-terminal cysteine residue to thioester-modified glass slides. [28][29][30] A more sophisticated reaction for oriented immobilization of peptide derivatives was introduced by Houseman et al. [31] A Diels–Alder reaction between benzoquinone groups on self-assembled monolayers and cyclopentadiene– peptide conjugates led to efficient covalent attachment of kinase substrate peptides that were efficiently phosphorylated by c-Src kinase. [31] The surface-bound peptides' accessibility to the proteins or enzymes used in screening has also been identified as a critical factor. ...
... –double peptide synthesized SPOTs containing the PTP-1B substrate IYETDYZRKGG (Z = phosphotyrosine) and on the second site a scan of 11-mer overlapping peptides derived from the cytoplasmic domain of insulin receptor –GST-fusion protein of substrate-trapping mutant of protein tyrosine phosphatase 1B (D181A), radioactively labeled by incubation with protein kinase A in the presence of [–double peptide synthesis of insulin receptor-derived substrate peptide IYETDYZRKGG (Z = phosphotyrosine) and binding motif for YAP WW1 domain and p53 binding protein-2 (YPPYPPPPYPS) PKA and Abl; Pro-Q Diamond phospho-specific stain or FITC-labeled antiphosphotyrosine antibody for detection kemptide, p60 c-src (521–533), delta sleep-inducing peptide (DSIP), phosphoDSIP, CamKII peptide (GS1–10), different proteins DIPP [52] GST-proteins of substrate-trapping mutants of tyrosine phosphatases (PTP-H1, SAP-1, TC-PTP, PTP-1B) radioactively labeled by incubation with protein kinase A in the presence of g 32 P-ATP 7 peptides (14-mers) derived from human GHR together with the appropriate phosphotyrosine-containing derivatives SPOT [64] Tyrosine kinase p60 c-scr and FITC-labeled antiphosphotyrosine antibody deletion, alanine-scanning, positional scanning, and full combinatorial mixture libraries of CGG-YIYGSFK (p60 c-src substrate) DIPP [29, 30] –protein kinase A and tyrosine kinase Abl and Cy5-labeled anti- bodies ...
Hundreds and thousands of simultaneous kinase assays are possible by using peptide arrays. In this minireview, we summarize production principles, library types, and assay strategies used for kinase profiling on peptide arrays. The second part gives a comprehensive review of the various peptide microarray applications in kinase research.
Speed and throughput are vital ingredients for discovery driven, “-omics” research. The small molecule microarray (SMM) succeeds at delivering phenomenal screening throughput and versatility. The concept at the heart of the technology is elegant, yet simple: by presenting large collections of molecules in high density on a flat surface, one is able to interrogate all possible interactions with desired targets, in just a single step. SMMs have become established as the choice platform for screening, lead discovery, and molecular characterization. This introduction describes the principles governing microarray construction and use, focusing on practical challenges faced when conducting SMM experiments. It will explain the key design considerations and lay the foundation for the chapters that follow. (An earlier version of this chapter appeared in Small Molecule Microarrays: Methods and Protocols, published in 2010.)
On a past volume of this monograph we have reviewed general aspects of the varied technologies available to generate peptide arrays. Hallmarks in the development of the technology and a main sketch of preparative steps and applications in binding assays were used to walk the reader through details of peptide arrays. In this occasion, we resume from that work and bring in some considerations on quantitative evaluation of measurements as well as on selected reports applying the technology.
Applications of peptide arrays in the field of enzyme profiling are described in detail. Focus is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimization for kinases. Additionally, several examples for reliable identification of substrates for other enzymes like lysine methyl transferases and ATP-ribosyltransferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological fluids like cell lysates or lysates of complete organisms will be analysed. All published examples of peptide arrays used for enzyme profiling are summarized comprehensively.
Protein microarrays allow for the parallel analysis of a large number of proteins with respect to their abundance or activity and, therefore, are playing an emerging role in proteome research.Protein expression profiling, detection of enzymatic activity and protein interaction mapping are the most important applications of protein microarrays. The biological content, as well as the detection strategies, vary significantly between these applications, making the protein microarray technologies much more varied than those used with DNA microarrays.In most cases, the biological content on the microarray, as well as the analytes, are proteins, which, due to their delicate nature and the heterogeneity of their properties, complicate the manufacturing and assay design. The potential advantages of protein microarrays over nonparallel, macroscopic technologies cannot, however, be overestimated.Keywords:Capture Agents;Enzyme Activity Profiling;Multiplexed Protein Assay;Protein Expression Profiling;Protein Interaction Profiling;Protein Microarray
