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Identification of the Molecular Composition of the 20S Proteasome of Mouse Intestine by High-Resolution Mass Spectrometric Proteome AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In the last years, intracellular protein degradation by the proteasome has become a focus area of scientific interest. Here, we describe a proteomics approach for the molecular mapping of the constituents of the proteolytically active core particle, the constitutive 20S proteasome from mouse intestine. In addition to the proteomics workflow widely used for protein isolation, gel electrophoretic separation, in-gel digestion, and UV-MALDI mass spectrometry, high-resolution Fourier transform ion cyclotron resonance mass spectrometry using infrared-MALDI ionisation (IR-MALDI FTICR-MS) has been employed as an efficient method f...
Source: Springer protocols feed by Protein Science - September 9, 2009 Category: Biochemistry Source Type: info

Bioinformatical Approaches to Detect and Analyze Protein InteractionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Protein-protein interactions are the building blocks of cellular networks and at the heart of cellular regulation. However, their experimental identification is still a challenge. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Sequential Peptide Affinity Purification System for the Systematic Isolation and Identification of Protein Complexes from Escherichia coliEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichi...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Tandem Affinity Purification of Protein Complexes from Mammalian Cells by the Strep/FLAG (SF)-TAP TagEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled efficient and large-scale purification of native protein complexes. The SF-TAP tag, a modified version of the TAP tag, allows a fast and straightforward purification of protein complexes from mammalian cells. It consists of a tandem Strep-tag II and a FLAG epitope (SF-TAP). The SF-TAP tag allows a native elution of protein complexes without proteolytic cleavage needed in the original TAP procedure. Besides the SF-TAP protocol, the principal idea of a ...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Enrichment and Preparation of Plasma Membrane Proteins from Arabidopsis thaliana for Global Proteomic Analysis Using Liquid Chromatography-Tandem Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The plasma membrane proteins are critical components in cellular control and differentiation and thus are of special interest to those studying signal transduction mechanisms in all organisms. When conducting proteomic studies on membrane components of cells and tissues, the complexity is not simply confined to the large number of proteins present in the sample but also to the highly hydrophobic nature of membrane proteins containing multiple transmembrane domains. Consequently, these proteins are more difficult to analyze by mass spectrometry, particularly if protein sequence coverage is to be established. This chapter co...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Isolation of Plasma Membranes from the Nervous System by Countercurrent Distribution in Aqueous Polymer Two-Phase SystemsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The plasma membrane separates the cell-interior from the cell’s environment. To maintain homeostatic conditions and to enable transfer of information, the plasma membrane is equipped with a variety of different proteins such as transporters, channels, and receptors. The kind and number of plasma membrane proteins are a characteristic of each cell type. Owing to their location, plasma membrane proteins also represent a plethora of drug targets. Their importance has entailed many studies aiming at their proteomic identification and characterization. Therefore, protocols are required that enable their purification in hi...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Part VII Subcellular Proteomics Organelle Proteomics: Reduction of Sample Complexity by Enzymatic In-Gel Selection of Native ProteinsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
One major problem in proteomics is the biochemical complexity of living cells. Therefore, strategies are needed to reduce the number of proteins to a manageable amount, enabling researchers to make a statement concerning protein functions. One possibility is the isolation of organelles, which reduces the protein complexity, e.g., for the chloroplast to an estimated number of 2,700 different proteins. For further limitation of the protein number, proteins can be divided into membrane and soluble proteins, which can be analyzed separately in a subsequent step. For membrane proteins, blue native polyacrylamide gel electrophor...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Isotope-Labeling and Affinity Enrichment of Phosphopeptides for Proteomic Analysis Using Liquid Chromatography-Tandem Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The reversible phosphorylation of proteins is a dynamic process that plays a major role in many vital physiological processes by transmitting signals within cellular pathways and networks. Proteomic measurements using mass spectrometry are capable of characterizing the sites of protein phosphorylation and to quantify their change in abundance. However, the low stoichiometry of protein phosphorylation events often preclude mass spectrometry detection and require additional sample preparation steps to facilitate their characterization. Many analytical methods have been used to map and quantify changes in phosphorylation, and...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Proteomics Identification of Oxidatively Modified Proteins in BraEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Several studies demonstrated the involvement of free radicals in the pathophysiology of neurodegenerative diseases. Once formed, reactive oxygen species (ROS) can promote multiple forms of oxidative damage, including protein oxidation, and thereby influence the function of a diverse array of cellular processes leading inevitably to neuronal dysfunctions. Protein oxidation can therefore rapidly contribute to oxidative stress by directly affecting cell signaling, cell structure, and enzymatic processes such as metabolism. There are many different modes of inducing protein oxidation including metal-catalyzed oxidation, oxidat...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Shotgun Protein Identification and Quantification by Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Shotgun proteomics is based on identification and quantification of peptides from digested proteins using tandem mass spectrometry. In this chapter, we discuss computational methods to analyze tandem mass spectra of peptides, including database searching, de novo peptide sequencing, hybrid approaches, library searching, and unrestricted modification search. A special focus is given to database searching programs since they are most widely used. The process of inferring proteins from identified peptides is then discussed. We also provide description of key steps in the quantitative analysis of mass spectrometry proteomics d...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Algorithms and DatabasesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The capacity of proteomics methods and mass spectrometry instrumentation to generate data has grown substantially over the past years. This data volume growth has in turn led to an increased reliance on software to identify peptide or protein sequences from the recorded mass spectra. Diverse algorithms can be applied for the processing of these data, each performing a specific task such as spectrum quality filtering, spectral clustering and merging, assigning a sequence to a spectrum, and assessing the validity of these assignments. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Electrospray Mass Spectrometry for Quantitative Plasma Proteome AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Electrospray ionization mass spectrometry (ESI-MS) is an efficient soft ionization procedure for macro biomolecules. However, it is a rather delicate process to produce charged molecules for mass-to-charge ratio (m/z) based measurement. In this chapter, the mechanism of ESI is briefly presented, and the experimental pipeline for quantitative profiling of plasma proteins (prefractionation immunodepletion, protein isotope tagging, 2D-HPLC separation of intact proteins, and LC-MS) is presented as applied by our group in studies of cancer biomarker discovery. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

iTRAQ-Labeling of In-Gel Digested Proteins for Relative QuantificationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In addition to standard MS-based protein identification, quantification of proteins by mass spectrometry (MS) is rapidly gaining acceptance in proteomic studies. MS-based quantification involves either the incorporation of stable isotopes or can be performed label-free. Recently, more attention has been devoted to label-free quantification; however, this approach has not been fully established among the proteomic community yet. More common is still the introduction of stable isotopes, which can be done by metabolic (e.g., SILAC) or by chemical (e.g., ICAT, iTRAQ, etc.) labeling. Here, we present an overall quantification s...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Liquid Chromatography–Mass Spectrometry-Based Quantitative ProteomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
During the last decades, molecular sciences revolutionized biomedical research and gave rise to the biotechnology industry. During the next decades, the application of the quantitative sciences – informatics, physics, chemistry, and engineering – to biomedical research brings about the next revolution that will improve human healthcare and certainly create new technologies, since there is no doubt that small changes can have great effects. It is not a question of “yes” or “no,” but of “how much,” to make best use of the medical options we will have. (Source: Springer protocol...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Characterization of Platelet Proteins Using Peptide Centric ProteomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In modern proteomics, undersampling of low abundant, cumbersome, and hydrophobic proteins states one of the major problems. To overcome this, especially in two 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) eminent drawbacks, the so-called peptide-centric techniques have been developed. These approaches do not separate proteins prior to digestion, but instead proteolytically generate peptide mixtures after it. However, by this procedure already complex protein mixtures become even more extensive peptide mixtures. Particularly, when dealing with large proteomes, the generated sample complexity is vast and ther...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Multidimensional Protein Identification TechnologyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Over the past years, large-scale analysis of proteomes gained increased interest to obtain a fast but nevertheless comprehensive overview about cellular protein content. While a complete proteome cannot be covered using current technologies because of its enormous diversity, subfractionation to reduce the complexity has become mandatory. While 2D-PAGE is well established as a high-resolution protein separation technique, it suffers from drawbacks, which can be overcome by using peptide separation methods based on multidimensional liquid chromatography. One of these technologies is multidimensional protein identification te...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

A Newcomer’s Guide to Nano-Liquid-Chromatography of PeptidesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
LC-MS/MS is one of the most powerful techniques in the field of proteomics allowing high throughput identification of proteins out of complex protein mixtures. Besides high sample throughput, the analytical sensitivity is one of the major benefits of this technology. A prerequisite for sensitive LC-MS/MS approaches is chromatography with very low flow rates in the nanoliter per minute range, usually referred to as nano-liquid chromatography (nano-LC). However, to perform this separation technology, an appropriate instrumental setup as well experienced operators are a prerequisite. The aim of this chapter is to help nano-LC...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Capillary Electrophoresis Coupled to Mass Spectrometry for Proteomic Profiling of Human Urine and Biomarker DiscoveryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Currently, the main focus of clinical proteome analysis is on detection and identification of polypeptides that significantly change owing to pathological changes. Capillary electrophoresis coupled online to an electrospray ionization time of flight mass spectrometer (CE-MS) allows the differential display of a large number of polypeptides in a single, reproducible, and time-limited step and enables the comparison of different protein profiles for biomarker discovery. In addition to the reproducibility of the CE-MS setup, many further steps including data processing and mining, usage of biomarkers for diagnosis, and biomar...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Maldi msEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Matrix-assisted laser desorption/ionization (MALDI) is a key technique in mass spectrometry (MS)-based proteomics. MALDI MS is extremely sensitive, easy-to-apply, and relatively tolerant to contaminants. Its high-speed data acquisition and large-scale, off-line sample preparation has made it once again the focus for high-throughput proteomic analyses. These and other unique properties of MALDI offer new possibilities in applications such as rapid molecular profiling and imaging by MS. Proteomics and its employment in Systems Biology and other areas that require sensitive and high-throughput bioanalytical techniques greatly...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Identification of the Molecular Composition of the 2S Proteasome of Mouse Intestine by High-Resolution Mass Spectrometric Proteome AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In the last years, intracellular protein degradation by the proteasome has become a focus area of scientific interest. Here, we describe a proteomics approach for the molecular mapping of the constituents of the proteolytically active core particle, the constitutive 20S proteasome from mouse intestine. In addition to the proteomics workflow widely used for protein isolation, gel electrophoretic separation, in-gel digestion, and UV-MALDI mass spectrometry, high-resolution Fourier transform ion cyclotron resonance mass spectrometry using infrared-MALDI ionisation (IR-MALDI FTICR-MS) has been employed as an efficient method f...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Protein Detection and Quantitation Technologies for Gel-Based Proteome AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Numerous protein detection and quantitation methods for gel-based proteomics have been devised that can be classified in three major categories: (1) Universal (or “general”) detection techniques, which include staining with anionic dyes (e.g., Coomassie brilliant blue), reverse (or “negative”) staining with metal cations (e.g., imidazole-zinc), silver staining, fluorescent staining or labeling, and radiolabeling, (2) specific staining methods for the detection of post-translational modifications (e.g., glycosylation or phosphorylation), and (3) differential display techniques for the separation of m...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Non-classical 2-D ElectrophoresisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Classical 2-D electrophoresis (IEF/SDS 2-DE) using isoelectric focusing (IEF) and SDS-PAGE for the second dimension offers very high resolution for the separation of complex protein mixtures, but hydrophobic proteins can aggregate and are considerably under-represented in these 2-D gels. Non-classical 2-DE, as described here, summarizes several heterogeneous techniques, some of which, like BAC/SDS 2-DE and doubled SDS-polyacrylamide gel electrophoresis (dSDS-PAGE), intend to isolate the difficult hydrophobic proteins that are not accessible by classical 2-DE. Other types of non-classical 2-DE start with 1-D separation of n...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

High-Resolution Two-Dimensional ElectrophoresisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry is currently the workhorse for the majority of ongoing proteome projects. Although alternative/complementary technologies, such as MudPIT, ICAT, or protein arrays, have emerged recently, there is up to now no technology that matches 2-DE in its ability for routine parallel expression profiling of large sets of complex protein mixtures. 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. High-resolution...
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

Introduction to ProteomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this chapter, the evolvement of proteomics from classical protein chemistry is depicted. The challenges of complexity and dynamics led to several new approaches and to the firm belief that a valuable proteomics technique has to be quantitative. Protein-based vs. peptide-based techniques, gel-based vs. non-gel-based proteomics, targeted vs. general proteomics, isotopic labeling vs. label-free techniques, and the importance of informatics are summarized and compared. A short outlook into the near future is given at the end of the chapter. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - August 20, 2009 Category: Biochemistry Source Type: info

PASE: A Web-Based Platform for Peptide/Protein Microarray ExperimentsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Peptide microarray technology requires bioinformatics and statistical tools to manage, store, and analyze the large amount of data produced. To address these needs, we developed a system called protein array software environment (PASE) that provides an integrated framework to manage and analyze microarray information from polypeptide chip technologies. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Qualitative and Quantitative Analysis of Peptide Microarray Binding Experiments Using SVM-PEPARRAYEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A main objective of analyzing peptide array-based binding experiments is to uncover the relationship between a peptide sequence and the binding outcome. Limited by the peptide array technologies available for applications, few attempts have been made to construct qualitative or quantitative models that depict the peptide sequence:binding strength relationships in peptide microarray-based binding studies. There has been a long history of similar modeling efforts based on low-throughput binding data in the areas of T-cell epitope screening and kinase substrate mapping, however. The keen needs in peptide array applications an...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Web-Based Design of Peptide Microarrays Using μPepArray ProEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Peptide microarrays (peptide arrays) have increasingly become an important research tool for studying protein detection, profiling, and protein–protein interactions, and they have the potential to foster high-throughput protein analysis as DNA arrays did for genomics research a decade ago. Recently, technologies have emerged that allow flexible synthesis of high-density peptide arrays based on specific application needs (e.g., phosphopeptide microarrays). To fully unleash the power of this promising research tool, significant efforts are required to develop computational and informatics resources that facilitate the ...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Visualisation and Pre-processing of Peptide Microarray DataEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The data files produced by digitising peptide microarray images contain detailed information on the location, feature, response parameters and quality of each spot on each array. In this chapter, we will describe how such peptide microarray data can be read into the R statistical package and pre-processed in preparation for subsequent comparative or predictive analysis. We illustrate how the information in the data can be visualised using images and graphical displays that highlight the main features, enabling the quality of the data to be assessed and invalid data points to be identified and excluded. The log-ratio of the...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Profiling the Autoantibody Repertoire by Screening Phage-Displayed Human cDNA LibrariesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The advent of the serological identification of antigens by procedures such as cDNA cloning and recombinant protein expression has allowed the direct molecular definition of immunogenic proteins. The phage-display technology provides several advantages over conventional immunoscreening procedures based on plasmid or lambda-phage cDNA libraries. So far, attempts to display open reading frames, such as those encoded by cDNA fragments, on filamentous phages have not been very successful. We managed to develop a strategy based on “folding reporters” which allows filtering out open reading frames from DNA and displa...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

An Advanced Application of Protein Microarrays: Cell-Based Assays for Functional GenomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microarrays have become common tools for approaching different experimental questions: DNA, protein and peptide arrays offer the power of multiplexing the assay and by means of miniaturization technology, the possibility to reduce cost and amount of samples and reagents. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

The Peptide Microarray-Based Assay for Kinase Functionality and Inhibition StudyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report a microarray format for the detection of kinase functionality/inhibition based on marking peptide phosphorylation/biotinylation events by the attachment of gold nanoparticles followed by silver deposition for signal enhancement. The detection principle is resonance light scattering (RLS) or surface-enhanced Raman spectroscopy (SERS). α-Catalytic subunit of cyclic adenosine 5′-monophosphate (cAMP) dependent protein kinase (PKA) and its well-known substrate, kemptide, are used for the purpose of monitoring phosphorylation and inhibition. As expected, highly selective inhibition of PKA is demonstrated wi...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Polypyrrole–Peptide Microarray for Biomolecular Interaction Analysis by SPR ImagingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Nowadays, high-throughput analysis of biological events is a great challenge which could take benefit of the recent development of microarray devices. The great potential of such technology is related to the availability of a chip bearing a large set of probes, stable and easy to obtain, and suitable for ligand-binding detection. Here, we describe a new method based on polypyrrole chemistry, allowing the covalent immobilization of peptides in a microarray format and on a gold surface compatible with the use of surface plasmon resonance. This technique is then illustrated by the detection and characterization of antibodies ...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

A Novel Combinatorial Approach to High-Density Peptide ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Combinatorial synthesis of peptides on solid supports (1), either as spots on cellulose membranes (2) or with split-pool-libraries on polymer beads (3), substantially forwarded research in the field of peptide–protein interactions. Admittedly, these concepts have specific limitations, on one hand the number of synthesizable peptide sequences per area, on the other hand elaborate decoding/encoding strategies, false-positive results and sequence limitations. We recently established a method to produce high-density peptide arrays on microelectronic chips (4). Solid amino acid microparticles were charged by friction and ...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Self-Assembly of PNA-Encoded Peptides into MicroarraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Several technologies have been described to immobilize libraries of small molecules or peptides in a microarray format. Herein, we describe protocols for an alternative strategy whereby each small molecule or peptide within a library is labeled with a peptide nucleic acid (PNA) tag such that they self-assemble in a microarray format upon hybridization with readily available DNA arrays. An important asset of the method is that it allows the library to be used in solution prior to hybridizing and as such offers the opportunity to separate the inhibitors bound to the protein from the rest of the library. Two methods based on ...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Peptide Microarrays on Bisphenol A PolycarbonateEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe in particular the preparation of peptide microarrays on PC using semicarbazide-functionalized silica nanoparticles and in situ semicarbazone ligation with glyoxylyl-peptides. The microarrays were used for the detection of antibodies using fluorescence detection. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

High-Density Peptide Microarrays for Reliable Identification of Phosphorylation Sites and Upstream KinasesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The human genome encodes about 25,000 genes. This number seems to be very small compared to the multitude of different protein functions in highly regulated pathways that are responsible for complex biochemical mechanisms like growth, metabolism, signal transduction and reproduction. Obviously, there are mechanisms creating additional protein diversity. The most important mechanism is post-translational modification (PTM) changing protein surfaces by phosphorylation, sulfation, acetylation, methylation and sumoylation resulting in an about 100-fold higher complexity (1, 2). This chapter presents a very efficient way to det...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

CelluSpots™: A Reproducible Means of Making Peptide Arrays for the Determination of SH2 Domain Binding SpecificityEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Peptide arrays differ from conventional peptide synthesis in that hundreds upon thousands of peptides are synthesized and presented on a planar surface at a time. While direct synthesis of peptide arrays on a functionalized surface is feasible, reprinting of pre-made peptides offers flexibility and reproducibility and drastically reduces cost when multiple copies of the same or related peptide arrays are needed. Cellu-Spot™, a method developed by Intavis, opens a new route in peptide array synthesis and printing and overcomes certain limitations of the SPOT membrane. This technique was used to produce hundreds of pho...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Characterization of Kinase Target Phosphorylation Consensus Motifs Using Peptide SPOT ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The human proteome is known to contain >500 protein kinases, which regulate almost all facets of cellular biology by the post-translational attachment of a phosphate moiety to serine, threonine, or tyrosine residues within a substrate protein. Most protein kinases remain poorly characterized and, as a result, current studies are directed toward defining their target substrates experimentally to gain a comprehensive view of the signaling proteins and pathways modulated by these kinases. Herein, we describe a rapid and convenient method for elucidating the consensus substrate motif for phosphorylation by a protein kinase ...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Rapid Identification of Linear Protein Domain Binding Motifs Using Peptide SPOT ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Understanding protein–protein interactions is a key step in unravelling the roles proteins play in cellular function. The ability to analyse protein–protein interactions rapidly and economically is a powerful research tool. Using peptide SPOT arrays, peptides of known sequence can be synthesized directly in discrete spots on a cellulose membrane and assayed for an interaction with a protein of interest. Several hundred peptides can be synthesized on each cellulose membrane; therefore, this method is amenable to designing high-throughput peptide binding studies. SPOT arrays are particularly well suited for deduc...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Synthesis of Peptide Arrays Using SPOT-Technology and the CelluSpots-MethodEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Peptide synthesis on cellulose using the SPOT technology follows the standard Fmoc-chemistry and can be performed manually or automated. This method allows the synthesis of low-cost peptide arrays containing around 900 large spots of addressable peptides on a cellulose sheet of 19 cm × 29 cm. These peptides can be cleaved from the cellulose support by ammonia gas and afterward spotted on glass microchips. Alternatively, the peptides can be synthesized on modified cellulose discs and CelluSpot microarrays can be produced. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

A Designed Peptide Chip: Protein Fingerprinting Technology with a Dry Peptide Array and Statistical Data MiningEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
There has recently been increased interest in the potential for microarray technologies to study protein networks in a whole cell system within a single experiment. Protein-detecting microarrays are composed of numerous agents immobilized within a tiny area on solid surfaces to capture targeted proteins and to detect interactions in a high-throughput fashion. In this chapter, in order to extend the usability of peptide microarrays, we describe a novel dry peptide microarray format to obtain protein fingerprint (PFP) data sets and a statistical PFP data manipulation technique to quantitatively analyze targeted proteins. (So...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Mapping Functional Prion–Prion Protein Interaction Sites Using Prion Protein Based Peptide-ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Protein–protein interactions are at the basis of most if not all biological processes in living cells. Therefore, adapting existing techniques or developing new techniques to study interactions between proteins are of importance in elucidating which amino acid sequences contribute to these interactions. Such new insights may in turn lead to improved understanding of the processes underlying disease and possibly provide the basis for new therapeutic approaches. Here we describe the novel use of an ovine prion protein-based peptide-array normally used for determining prion-specific antibody epitopes, with the prospect ...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Antimicrobial Peptide Arrays for Detection of Inactivated Biothreat AgentsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Arrays of immobilized antimicrobial peptides are used to detect bacterial, viral, and rickettsial pathogens, including inactivated biothreat agents. These arrays differ from the many combinatorial peptide arrays described in the literature in that the peptides used here have naturally evolved to interact with and disrupt microbial membranes with high affinity but broad specificity. The interaction of these naturally occurring peptides with membranes of pathogens has been harnessed for the purpose of detection, with immobilized antimicrobial peptides acting as “capture” molecules in detection assays. Methods are...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Epitope Mapping of Human Chromogranin A by Peptide MicroarraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this chapter we report on the characterization of linear antigenic sites of human chromogranin A (CgA), a useful tissue and serum marker for neuroendocrine tumours and a precursor of many biologically active peptides. The epitope mapping of CgA has been carried out by peptide microarrays on glass slides coated by a copolymer of N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS) and [3-(methacryloyl-oxy) propyl] trimethoxysilyl (MAPS). The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substr...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Molecular Simulations of Peptides: A Useful Tool for the Development of New Drugs and for the Study of Molecular RecognitionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The study of the molecular recognition and self-organization properties of peptides has emerged in recent years as a very active and diverse field of research, ranging from biomedicine to biotechnology and even to material sciences. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Using Peptide Array to Identify Binding Motifs and Interaction Networks for Modular DomainsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Specific protein–protein interactions underlie all essential biological processes and form the basis of cellular signal transduction. The recognition of a short, linear peptide sequence in one protein by a modular domain in another represents a common theme of macromolecular recognition in cells, and the importance of this mode of protein–protein interaction is highlighted by the large number of peptide-binding domains encoded by the human genome. This phenomenon also provides a unique opportunity to identify protein–protein binding events using peptide arrays and complementary biochemical assays. Accordi...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Peptide Arrays for Enzyme ProfilingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Enzymes are key molecules in signal transduction pathways. However, only a small fraction of more than 500 predicted human kinases, 250 proteases and 250 phosphatases is characterized so far. Peptide microarray-based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. Additionally, patterns of enzymatic activities could be used to fingerprint the status of cells or organisms. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimization of enzyme substrates. A comprehensive overv...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

Exploring and Profiling Protein Function with Peptide ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Development of array technologies started in the late 1980s and was first extensively applied to DNA arrays especially in the genomic field. Today this technique has become a powerful tool for high-throughput approaches in biology and chemistry. Progresses were mainly driven by the human genome project and were associated with the development of several new technologies, which led to the onset of additional “omic” topics like proteomics, glycomics, antibodyomics or lipidomics. The main characteristics of the array technology are (i) spatially addressable immobilization of a huge number of different capture mole...
Source: Springer protocols feed by Protein Science - July 31, 2009 Category: Biochemistry Source Type: info

ArrayTrack: An FDA and Public Genomic ToolEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A robust bioinformatics capability is widely acknowledged as central to realizing the promises of toxicogenomics. Successful application of toxicogenomic approaches, such as DNA microarrays, inextricably relies on appropriate data management, the ability to extract knowledge from massive amounts of data, and the availability of functional information for data interpretation. At the FDA’s National Center for Toxicological Research (NCTR), we are developing a public microarray data management and analysis software, called ArrayTrack, that is also used in the routine review of genomic data submitted to the FDA. ArrayTra...
Source: Springer protocols feed by Protein Science - June 30, 2009 Category: Biochemistry Source Type: info

Translational Research and Biomedical InformaticsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A critical need exists to address real issues that appear when a physician is faced with a patient and the need to make clinical decisions that will impact the patient, their quality life, and those of the patient’s family. Bridging this gap between the clinical need and the available technologies, clinical data, and clinician input is the role that Biomedical Informatics can play in driving the evolution of patient care in the post-genome era. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - June 30, 2009 Category: Biochemistry Source Type: info