MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Protein Science' source. Sat, 30 Jan 2010 16:09:19 +0100 http://www.medworm.com/index.php?rid=2777777&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_10 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 for protein identification by peptide mass fingerprint. The 20S proteasome subunits α1–α7 and β1–β7 were completely and unambiguously ide... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2777777 Wed, 09 Sep 2009 18:00:17 +0100 2777777 http://www.medworm.com/index.php?rid=2715405&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_23 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715405 Thu, 20 Aug 2009 11:31:53 +0100 2715405 http://www.medworm.com/index.php?rid=2715404&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_22 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 Escherichia coli protein complexes (J Proteome Res 3:463–468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715404 Thu, 20 Aug 2009 11:31:53 +0100 2715404 http://www.medworm.com/index.php?rid=2715403&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_21 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 pathway mapping by subsequent tagging of copurified proteins is demonstrated for the interactome of the MAPKKK Raf. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715403 Thu, 20 Aug 2009 11:31:53 +0100 2715403 http://www.medworm.com/index.php?rid=2715402&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_20 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 contains a method for extraction, solubilization, alkylation, proteolysis, and identification of hydrophobic integral plasma membrane proteins for large-scale proteomic ana... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715402 Thu, 20 Aug 2009 11:31:53 +0100 2715402 http://www.medworm.com/index.php?rid=2715401&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_19 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 high purity and quantity. Here, we report a protocol, based on aqueous polymer two-phase systems, which fulfils these demands. Furthermore, the protocol is time-saving and ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715401 Thu, 20 Aug 2009 11:31:53 +0100 2715401 http://www.medworm.com/index.php?rid=2715400&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_18 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 electrophoresis (BN-PAGE) in combination with enzymatic in-gel assays (e.g. detection of NADPH dehydrogenases) is a suitable method for a fast and easy visualization and identificat... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715400 Thu, 20 Aug 2009 11:31:53 +0100 2715400 http://www.medworm.com/index.php?rid=2715399&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_17 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 this chapter will present two methods that can be used for extraction of phosphopeptides from protein and proteome digests to map phosphorylation sites using liquid chro... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715399 Thu, 20 Aug 2009 11:31:53 +0100 2715399 http://www.medworm.com/index.php?rid=2715398&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_16 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, oxidation-induced cleavage of peptide chain, amino acid oxidation, and the covalent binding of lipid peroxidation products or advanced glycation end proteomics.In this paper we... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715398 Thu, 20 Aug 2009 11:31:53 +0100 2715398 http://www.medworm.com/index.php?rid=2715397&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_15 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 data. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715397 Thu, 20 Aug 2009 11:31:52 +0100 2715397 http://www.medworm.com/index.php?rid=2715396&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_14 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715396 Thu, 20 Aug 2009 11:31:52 +0100 2715396 http://www.medworm.com/index.php?rid=2715395&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_13 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715395 Thu, 20 Aug 2009 11:31:52 +0100 2715395 http://www.medworm.com/index.php?rid=2715394&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_12 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 strategy for chemical labeling of in-gel digested proteins using iTRAQ reagents. This includes (1) protein separation by gel electrophoresis, (2) excision of protein bands... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715394 Thu, 20 Aug 2009 11:31:52 +0100 2715394 http://www.medworm.com/index.php?rid=2715393&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_11 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 protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715393 Thu, 20 Aug 2009 11:31:52 +0100 2715393 http://www.medworm.com/index.php?rid=2715392&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_9 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 therefore difficult to analyze. When separated and analyzed by LC/MS, too many peptides may enter the mass spectrometer at a certain time point, and only a small fraction of ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715392 Thu, 20 Aug 2009 11:31:52 +0100 2715392 http://www.medworm.com/index.php?rid=2715391&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_8 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 technology (MudPIT). It consists of two orthogonal separation systems – strong cation exchange (SCX) and reversed phase (RP) – coupled online in an automated fa... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715391 Thu, 20 Aug 2009 11:31:52 +0100 2715391 http://www.medworm.com/index.php?rid=2715390&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_7 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 newcomers to get introduced to this fascinating technology. Technical components of nano-LC systems like solvent delivery systems, sample injection systems, and nano-chr... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715390 Thu, 20 Aug 2009 11:31:52 +0100 2715390 http://www.medworm.com/index.php?rid=2715389&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_6 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 biomarker sequencing are necessary to answer the demands of biomarker discovery of clinical significance. In this chapter, we discuss materials and methods for CE-MS-based clin... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715389 Thu, 20 Aug 2009 11:31:52 +0100 2715389 http://www.medworm.com/index.php?rid=2715388&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_5 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 depend on these methodologies. This chapter provides a basic introduction to the MALDI methodology and its general application in proteomic research. It describes the ba... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715388 Thu, 20 Aug 2009 11:31:52 +0100 2715388 http://www.medworm.com/index.php?rid=2715387&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_10 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 for protein identification by peptide mass fingerprint. The 20S proteasome subunits α1–α7 and β1–β7 were completely and unambiguously ide... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715387 Thu, 20 Aug 2009 11:31:52 +0100 2715387 http://www.medworm.com/index.php?rid=2715386&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_4 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 multiple, covalently tagged samples in a single two-dimensional electrophoresis (2-DE) gel, followed by consecutive and independent visualization of these proteins to mini... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715386 Thu, 20 Aug 2009 11:31:52 +0100 2715386 http://www.medworm.com/index.php?rid=2715385&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_3 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 native proteins and complexes, like blue-native electrophoresis (BNE), clear-native electrophoresis (CNE), and high-resolution clear-native electrophoresis (hrCNE). These ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715385 Thu, 20 Aug 2009 11:31:52 +0100 2715385 http://www.medworm.com/index.php?rid=2715384&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_2 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 2-DE can resolve up to 5,000 proteins simultaneously (∼2,000 proteins routinely), and detect and quantify <1 ng of protein per spot. Today’s 2-DE technology... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715384 Thu, 20 Aug 2009 11:31:52 +0100 2715384 http://www.medworm.com/index.php?rid=2715383&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-157-8_1 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2715383 Thu, 20 Aug 2009 11:31:52 +0100 2715383 http://www.medworm.com/index.php?rid=2698900&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_24 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698900 Fri, 31 Jul 2009 23:00:00 +0100 2698900 http://www.medworm.com/index.php?rid=2698899&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_23 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 and the success of the modeling efforts in related fields have prompted us to develop SVM-PEPARRAY, a Web-based program capable of constructing qualitative and quantitative... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698899 Fri, 31 Jul 2009 23:00:00 +0100 2698899 http://www.medworm.com/index.php?rid=2698898&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_22 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 experimental design and data analysis for a wide range of peptide array-based applications. The design of peptide arrays is inherently more complex than that of DNA array... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698898 Fri, 31 Jul 2009 23:00:00 +0100 2698898 http://www.medworm.com/index.php?rid=2698897&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_21 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 foreground to background signal is used as a response index. Negative control responses serve as a reference against which “detectable” responses can be defi... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698897 Fri, 31 Jul 2009 23:00:00 +0100 2698897 http://www.medworm.com/index.php?rid=2698896&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_20 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 displaying them on filamentous phages in such a way that they are amenable to subsequent selection or screening. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698896 Fri, 31 Jul 2009 23:00:00 +0100 2698896 http://www.medworm.com/index.php?rid=2698895&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_19 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698895 Fri, 31 Jul 2009 23:00:00 +0100 2698895 http://www.medworm.com/index.php?rid=2698894&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_18 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 with the four inhibitors: H89, HA1077, mallotoxin and KN62. Furthermore, inhibition assay with inhibitors demonstrate the ability to detect kinase inhibition as well as der... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698894 Fri, 31 Jul 2009 23:00:00 +0100 2698894 http://www.medworm.com/index.php?rid=2698893&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_17 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 induced by hepatitis C virus and present in patients’ serums. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698893 Fri, 31 Jul 2009 23:00:00 +0100 2698893 http://www.medworm.com/index.php?rid=2698892&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_16 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 transferred to defined pixel electrodes onto the chip’s surface, where they couple to a functional polymer coating simply upon melting (Fig. 16.1 A–D,... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698892 Fri, 31 Jul 2009 23:00:00 +0100 2698892 http://www.medworm.com/index.php?rid=2698891&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_15 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 size exclusion filtration or gel electrophoresis to separate protein-bound inhibitors from the remaining library are described. (Source: Springer protocols feed by Protei... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698891 Fri, 31 Jul 2009 23:00:00 +0100 2698891 http://www.medworm.com/index.php?rid=2698890&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_14 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698890 Fri, 31 Jul 2009 23:00:00 +0100 2698890 http://www.medworm.com/index.php?rid=2698889&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_9 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 detect potential phosphorylation sites in proteins using overlapping peptide scans immobilized on glass slides. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698889 Fri, 31 Jul 2009 23:00:00 +0100 2698889 http://www.medworm.com/index.php?rid=2698888&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_8 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 phosphotyrosine-oriented peptide array libraries for determining the specificity of the Src homology 2 (SH2) domain. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698888 Fri, 31 Jul 2009 23:00:00 +0100 2698888 http://www.medworm.com/index.php?rid=2698887&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_7 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 using peptide SPOT arrays that are custom-synthesized on a cellulose membrane support. The definition of the target consensus motif provides an important starting point f... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698887 Fri, 31 Jul 2009 23:00:00 +0100 2698887 http://www.medworm.com/index.php?rid=2698886&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_6 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 deducing peptidic binding motifs within proteins that are difficult to purify in sufficient quantities for traditional biochemical analyses, as well as for determining binding... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698886 Fri, 31 Jul 2009 23:00:00 +0100 2698886 http://www.medworm.com/index.php?rid=2698885&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_5 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698885 Fri, 31 Jul 2009 23:00:00 +0100 2698885 http://www.medworm.com/index.php?rid=2698884&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_13 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. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698884 Fri, 31 Jul 2009 23:00:00 +0100 2698884 http://www.medworm.com/index.php?rid=2698883&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_12 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 that this would yield information on interaction sites between its PrP moiety and the ovine prion protein derived linear peptides. This adapted application of the peptide... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698883 Fri, 31 Jul 2009 23:00:00 +0100 2698883 http://www.medworm.com/index.php?rid=2698882&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_11 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 presented for immobilizing the antimicrobial peptides in planar arrays, performing direct and sandwich assays, and detecting bound targets. (Source: Springer protocols f... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698882 Fri, 31 Jul 2009 23:00:00 +0100 2698882 http://www.medworm.com/index.php?rid=2698881&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_10 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 substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which, despite peptide random orientation, linear epitopes are freely exposed. The res... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698881 Fri, 31 Jul 2009 23:00:00 +0100 2698881 http://www.medworm.com/index.php?rid=2698880&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_4 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698880 Fri, 31 Jul 2009 23:00:00 +0100 2698880 http://www.medworm.com/index.php?rid=2698879&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_3 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. Accordingly, high-density peptide array has emerged as a useful tool by which to map domain-mediated protein–protein interaction networks at the proteome level. Using the ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698879 Fri, 31 Jul 2009 23:00:00 +0100 2698879 http://www.medworm.com/index.php?rid=2698878&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_2 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 overview regarding enzyme profiling using peptide microarrays is presented with special focus on assay principles. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698878 Fri, 31 Jul 2009 23:00:00 +0100 2698878 http://www.medworm.com/index.php?rid=2698877&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-394-7_1 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 molecules; (ii) probing the array in a simultaneous and highly parallel manner with a biological sample; (iii) tendency towards miniaturization of the arrays; and (iv) softwa... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2698877 Fri, 31 Jul 2009 23:00:00 +0100 2698877 http://www.medworm.com/index.php?rid=2601241&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_20 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. ArrayTrack stores a full range of information related to DNA microarrays and clinical and non-clinical studies as well as the digested data derived from proteomics and metabonomi... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601241 Tue, 30 Jun 2009 23:00:00 +0100 2601241 http://www.medworm.com/index.php?rid=2601240&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_19 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) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601240 Tue, 30 Jun 2009 23:00:00 +0100 2601240 http://www.medworm.com/index.php?rid=2601239&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_18 MetaMiner (CF) is a data analysis platform for a broad range of CF researchers including wet lab biologists, bioinformaticians, clinicians, and chemists. To understand disease mechanisms and gain insight into complex biological actions, analysis of even simple gene interactions often requires integration of a variety of separate data resources such as literature, 3D molecular models, metabolic pathways, ontologies, small molecules, and drugs. Large-scale data sets from high-throughput screening assays, microarrays, and other data intensive procedures present an even greater challenge in data handling and analysis which now requires interdisciplinary teams of scientists with strikingly diverse backgrounds including computer scientists, statisticians, biologists, and clinicians. To address t... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601239 Tue, 30 Jun 2009 23:00:00 +0100 2601239 http://www.medworm.com/index.php?rid=2601238&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_17 In eukaryotes, besides alternative splicing and promoter regulation of “classical” genes, there is also another level of genetic regulation based on non-coding RNAs (ncRNAs). The most famous group of ncRNAs is microRNAs, probably the biggest number of genome regulators. Here, we summarize the knowledge that has been accumulated about the microRNA field, focusing our attention on brief history, biogenesis, regulated mechanism, computational methods of miRNA finding and miRNA target sites, miRNAs and diseases, and miRNAs and network analysis. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601238 Tue, 30 Jun 2009 23:00:00 +0100 2601238 http://www.medworm.com/index.php?rid=2601237&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_16 Network and pathway analysis tools are traditionally used to interrogate gene expression data in order to understand the biological processes affected by a particular manipulation or disease/condition of interest. A systems-level understanding of the biological processes affected in particular disease states can allow one to identify candidates not only for pharmaceutical intervention but also for potential prognostic and diagnostic markers for the disease. However, network and pathway analyses are currently underutilized in the interpretation of large-scale genetic association study results. While simple monogenic, overtly Mendelian diseases are easily understood in the context of a single genetic aberration, the vast majority of diseases follow more complex patterns of inheritance and ar... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601237 Tue, 30 Jun 2009 23:00:00 +0100 2601237 http://www.medworm.com/index.php?rid=2601236&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_15 High-throughput RNA interference (HT-RNAi) is a powerful research tool for parallel, ‘genome-wide’, targeted knockdown of specific gene products. Such perturbation of gene product expression allows for the systematic query of gene function. The phenotypic results can be monitored by assaying for specific alterations in molecular and cellular endpoints, such as promoter activation, cell proliferation and survival. RNAi profiling may also be coupled with drug screening to identify molecular correlates of drug response. As with other genomic-scale data, methods of data analysis are required to handle the unique aspects of data normalization and statistical processing. In addition, novel techniques or knowledge-mining strategies are required to extract useful biological information... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601236 Tue, 30 Jun 2009 23:00:00 +0100 2601236 http://www.medworm.com/index.php?rid=2601235&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_9 Recent advances in biotechnology have produced a wealth of genomic data, which capture a variety of complementary cellular features. While these data promise to yield key insights into molecular biology, much of the available information remains underutilized because of the lack of scalable approaches for integrating signals across large, diverse data sets. A proper framework for capturing these numerous snapshots of complementary phenomena under a variety of conditions can provide the holistic view necessary for developing precise systems-level hypotheses. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601235 Tue, 30 Jun 2009 23:00:00 +0100 2601235 http://www.medworm.com/index.php?rid=2601234&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_8 Prioritization, or ranking, of gene lists is becoming increasingly important for analyzing data generated from high-throughput assays like expression profiling and RNAi-based screening. This is especially the case when specific genes in a list need to be further validated using low-throughput experiments. In addition to gene set overlap enrichment methods, a complementary approach is to examine molecular interaction networks. These can provide putative functional insights based on gene connectivity, especially when many genes contain little or no annotation. For bench and computational biologists alike, using networks requires an informed selection of interaction data for network construction and strategies for managing network complexity. Moreover, graph theory and social network analysis... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601234 Tue, 30 Jun 2009 23:00:00 +0100 2601234 http://www.medworm.com/index.php?rid=2601233&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_7 The availability of whole genome sequences from various model organisms and increasing experimental data and literatures stimulated the evolution of a systems approach for biological research. The development of computational tools and algorithms to study biological pathway networks has made great progress in helping analyze research data. Pathway databases become an integral part of such an approach. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601233 Tue, 30 Jun 2009 23:00:00 +0100 2601233 http://www.medworm.com/index.php?rid=2601232&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_6 Set enrichment analytical methods have become commonplace tools applied to the analysis and interpretation of biological data. The statistical techniques are used to identify categorical biases within lists of genes, proteins, or metabolites. The goal is to discover the shared functions or properties of the biological items represented within the lists. Application of these methods can provide great biological insight, including the discovery of participation in the same biological activity or pathway, shared interacting genes or regulators, common cellular compartmentalization, or association with disease. The methods require ordered or unordered lists of biological items as input, understanding of the reference set from which the lists were selected, categorical classifiers describing th... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601232 Tue, 30 Jun 2009 23:00:00 +0100 2601232 http://www.medworm.com/index.php?rid=2601231&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_14 Analytical workflow technology, sometimes also called data pipelining, is the fundamental component that provides the scalable analytical middleware that can be used to enable the rapid building and deployment of an analytical application. Analytical workflows enable researchers, analysts and informaticians to integrate and access data and tools from structured and non-structured data sources so that analytics can bridge different silos of information; compose multiple analytical methods and data transformations without coding; rapidly develop applications and solutions by visually constructing analytical workflows that are easy to revise should the requirements change; access domain-specific extensions for specific projects or areas, for example, text extraction, visualisation, rep... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601231 Tue, 30 Jun 2009 23:00:00 +0100 2601231 http://www.medworm.com/index.php?rid=2601230&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_13 The vast quantities of information generated by academic and industrial research groups are reflected in a rapidly growing body of scientific literature and exponentially expanding resources of formalized data including experimental data from “-omics” platforms, phenotype information, and clinical data. For bioinformatics, several challenges remain: to structure this information as biological networks enabling scientists to identify relevant information; to integrate this information as specific “knowledge bases”; and to formalize this knowledge across multiple scientific domains to facilitate hypothesis generation and validation and, thus, the generation of new knowledge. Risk management in drug discovery and clinical research is used as a typical example to illust... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601230 Tue, 30 Jun 2009 23:00:00 +0100 2601230 http://www.medworm.com/index.php?rid=2601229&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_12 Cytoscape is a general network visualization, data integration, and analysis software package. Its development and use has been focused on the modeling requirements of systems biology, though it has been used in other fields. Cytoscape’s flexibility has encouraged many users to adopt it and adapt it to their own research by using the plugin framework offered to specialize data analysis, data integration, or visualization. Plugins represent collections of community-contributed functionality and can be used to dynamically extend Cytoscape functionality. This community of users and developers has worked together since Cytoscape’s initial release to improve the basic project through contributions to the core code and public offerings of plugin modules. (Source: Springer protocols f... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601229 Tue, 30 Jun 2009 23:00:00 +0100 2601229 http://www.medworm.com/index.php?rid=2601228&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_11 The metabolic networks are the most well-studied biochemical systems, with an abundance of in vitro and in vivo data available for quantitative estimation of its kinetic parameters. In this chapter, we present our approach to developing mathematical description of metabolic pathways. The model-based integration of reaction kinetics and the utilization of different types of experimental data including temporal dependencies have been described in detail. Software package DBSolve7 which allows us to develop kinetic model of the biochemical system and integrate experimental data has been presented. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601228 Tue, 30 Jun 2009 23:00:00 +0100 2601228 http://www.medworm.com/index.php?rid=2601227&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_10 Analysis of microarray, SNPs, proteomics, and other high-throughput (OMICs) data is challenging because of its biological complexity and high level of technical and biological noise. One way to deal with both problems is to perform analysis with a high-fidelity annotated knowledge base of protein interactions, pathways, and functional ontologies. This knowledge base has to be structured in a computer-readable format and must include software tools for managing experimental data, analysis, and reporting. Here we present MetaDiscovery, an integrated platform for functional data analysis which is being developed at GeneGo for the past 8 years. On the content side, MetaDiscovery encompasses a comprehensive database of protein interactions of different types, pathways, network models and 10 fun... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601227 Tue, 30 Jun 2009 23:00:00 +0100 2601227 http://www.medworm.com/index.php?rid=2601226&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_5 Protein interactions are the basic building blocks for assembly of pathways and networks. Almost any biologically meaningful functionality (for instance, linear signaling pathways, chains of metabolic reactions, transcription factor dimmers, protein complexes of transcriptosome, gene–disease associations) can be represented as a combination of binary relationships between “network objects” (genes, proteins, RNA species, bioactive compounds). Naturally, the assembled pathways and networks are only as good as their “weakest” link (i.e., a wrongly assigned interaction), and the errors multiply in multi-step pathways. Therefore, the utility of “systems biology” is fundamentally dependent on quality and relevance of protein interactions. The second impo... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601226 Tue, 30 Jun 2009 23:00:00 +0100 2601226 http://www.medworm.com/index.php?rid=2601225&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_4 The word protein is derived from the Greek “prota” meaning “of primary importance”, a designation which appropriately acknowledges the central role proteins play in biological systems. Following translation and folding into a remarkable array of three-dimensional structures, individual proteins achieve added complexity and functionality through the addition of modifications including glycosylation, acetylation, methylation, and phosphorylation. This complexity is further expanded through the non-covalent interactions that occur between proteins, and it is these interactions that form the foundation for many of the exquisitely regulated cellular processes essential to life. As a result, protein–protein interactions comprise an important class of targets for dru... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601225 Tue, 30 Jun 2009 23:00:00 +0100 2601225 http://www.medworm.com/index.php?rid=2601224&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_3 The past decade has seen a significant emergence in the availability and use of pathway analysis tools. The workflow that is supported by most of the pathway analysis tools is limited to either of the following: a. a network of genes based on the input data set, or b. the resultant network filtered down by a few criteria such as (but not limited to) i. disease association of the genes in the network; ii. targets known to be the target of one or more launched drugs; iii. ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601224 Tue, 30 Jun 2009 23:00:00 +0100 2601224 http://www.medworm.com/index.php?rid=2601223&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_2 We present a uniform methodology for estimating specificity, total number of binding sites, and sensitivity of data sets detected by these ChIP-based genome-wide experimental systems. We demonstrate strong heterogeneity of specific TF–DNA binding sites in terms of their avidity and by correlation between observed relative binding avidity of specific TF–DNA binding site and the level of mRNA transcription of the nearest gene target. Finally, we conclude that the sensitivity problem has not been resolved by current ChIP-based methods, including ChIP-Seq. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601223 Tue, 30 Jun 2009 23:00:00 +0100 2601223 http://www.medworm.com/index.php?rid=2601222&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-175-2_1 Natural language processing (NLP) technology can be used to rapidly extract protein–protein interactions from large collections of published literature. In this chapter we will work through a case study using MEDLINE® biomedical abstracts (1) to find how a specific set of 50 genes interact with each other. We will show what steps are required to achieve this using the I2E software from Linguamatics ( www.linguamatics.com (2)). (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2601222 Tue, 30 Jun 2009 23:00:00 +0100 2601222 http://www.medworm.com/index.php?rid=2223949&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_9 Establishing the phosphorylation pattern of proteins in a comprehensive fashion is an important goal of a majority of cell signaling projects. Phosphoproteomic strategies should be designed in such a manner as to identify sites of phosphorylation as well as to provide quantitative information about the extent of phosphorylation at the sites. In this chapter, we describe an experimental strategy that outlines such an approach using stable isotope labeling with amino acids in cell culture (SILAC) coupled to LC-MS/MS. We highlight the importance of quantitative strategies in signal transduction as a platform for a systematic and global elucidation of biological processes. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223949 Fri, 30 Jan 2009 05:00:00 +0100 2223949 http://www.medworm.com/index.php?rid=2223948&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_8 Mass spectrometry-based protein phosphorylation analysis on a proteome-wide scale remains a formida ble challenge, hampered by the complexity and dynamic range of protein expression on the global level and multi-site phosphorylation at substoichiometric ratios at the individual protein level. It is recognized that reduction of sample complexity or enrichment of the phosphopeptide pool is a necessary prereq uisite for global phospho-proteomics. Immobilized metal affinity chromatography (IMAC) and strong cation exchange chromatography, either alone or in tandem, have emerged as the most widely used chromatographic-based enrichment strategies. However, each is not without shortcomings. Both tech niques provide little fractionation of phosphorylated species and are compromised by competition a... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223948 Fri, 30 Jan 2009 05:00:00 +0100 2223948 http://www.medworm.com/index.php?rid=2223947&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_7 In recent years, stable isotope labeling by amino acids in cell culture (SILAC) has become increasingly popular as a quantitative proteomic method. In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing normal and heavy isotope amino acids. This makes proteins from the light and heavy cells distinguishable by mass spectrometry (MS) after the cell lysates are mixed and the proteins separated and/or enriched. SILAC is a powerful tool for the study of intracellular signal transduction. In particular, it has been very popular and successful in quantitative analysis of phosphoty-rosine (pTyr) proteomes to characterize pTyr-dependent signaling pathways. In this chapter, we describe the SILAC procedure and use EphB signaling pathway as an example to illust... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223947 Fri, 30 Jan 2009 05:00:00 +0100 2223947 http://www.medworm.com/index.php?rid=2223946&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_6 Phospho-proteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., immobilized metal affinity chromatography and titanium dioxide chromatography, provide varying degrees of selectivity and specificity for phosphopeptide enrichment. Furthermore, the number of multiply phosphorylated peptides that are identified in most published studies is rather low. Here the protocol for a new strategy that separates mono-phosphorylated pep-tides from multiply phosphorylated peptides using sequential elution from immobilized metal affinity chromatography is described. The two separate phosphopeptide fractions are subsequently analyzed by mass sp... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223946 Fri, 30 Jan 2009 05:00:00 +0100 2223946 http://www.medworm.com/index.php?rid=2223945&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_5 Titanium dioxide has very high affinity for phosphopeptides and it has become an efficient alternative to already existing methods for phosphopeptide enrichment from complex samples. Peptide loading in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding from nonphosphorylated peptides. The enriched phosphopeptides bound to the titanium dioxide are subsequently eluted from the micro-column using an alkaline buffer. Titanium dioxide chromatography is extremely tolerant towards most buffers used in biological experiments. It is highly robust and as such it has become one of the methods of choice in large-scale phospho-proteomics. Here we describe the p... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223945 Fri, 30 Jan 2009 05:00:00 +0100 2223945 http://www.medworm.com/index.php?rid=2223944&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_4 The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+;, Ga3+, and Al3+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphopeptide enrichment by IMAC is sensititive and specific for peptide mixtures derived from pure proteins or simple protein mixtures. The selectivity of the IMAC method is, however, limited when working with peptide mixtures derived from highly complex samples, e.g., whole-cell extracts, where sample prefractionation is advisable. Furthermore, lowering the pH value of the sample loading buffe... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223944 Fri, 30 Jan 2009 05:00:00 +0100 2223944 http://www.medworm.com/index.php?rid=2223943&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_3 Oxidative stress is the result of an increased presence of reactive oxygen species (ROS) in cells and is able to promote, among others, protein and lipid oxidation, DNA damage, mutagenesis, oncogenic activation, or inhibition of tumour suppression, resulting in pathological processes such as myocardial dysfunction or carcinogenesis. External treatment of cells with oxidants such as H2O2 or high intracellular levels of ROS has been shown to trigger protein tyrosine phosphorylation. This occurs, at least in part, through the oxidation of reactive cysteine groups in protein tyrosine phosphatases resulting in an inhibition of their activities. Herein, we focus on the characterization of stress-induced protein tyrosine phosphorylation events in a cellular model of human mammary luminal epitheli... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223943 Fri, 30 Jan 2009 05:00:00 +0100 2223943 http://www.medworm.com/index.php?rid=2223942&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_23 Protein phosphorylation is a widespread cellular process, and simplistic linear pathway models of kinase signaling likely under-represent the complexity of in vivo pathways. The recent massive increase in information available through protein interaction databases now allows construction of in silico models of protein networks that are underpinned by evidence from real biological systems. By combining protein phos-phorylation data with current databases of protein–protein and kinase–substrate interactions, sophisticated models of intracellular protein phosphorylation signaling can be constructed for a system of interest. The kinase interaction network can be visualized, analyzed by graph theory, and investigated for hypotheses that are not otherwise obvious. (Source: Springer p... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223942 Fri, 30 Jan 2009 05:00:00 +0100 2223942 http://www.medworm.com/index.php?rid=2223941&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_22 As extensive mass spectrometry-based mapping of the phosphoproteome progresses, computational anal ysis of phosphorylation-dependent signaling becomes increasingly important. The linear sequence motifs that surround phosphorylated residues have successfully been used to characterize kinase–substrate spe cificity. Here, we briefly describe the available resources for predicting kinase-specific phosphorylation from sequence properties. We address the strengths and weaknesses of these resources, which are based on methods ranging from simple consensus patterns to more advanced machine-learning algorithms. Furthermore, a protocol for the use of the artificial neural network based predictors, NetPhos and NetPhosK, is provided. Finally, we point to possible developments with the intention ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223941 Fri, 30 Jan 2009 05:00:00 +0100 2223941 http://www.medworm.com/index.php?rid=2223940&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_21 Protein modifications such as phosphorylation are often studied by two-dimensional gel electrophoresis, since the perturbation in the protein's pI value is readily detected by this method. It is important to be able to calculate the changes in the pI values that specific post-translational modifications cause and to visualize how these changes will effect protein migration on 2D gels. To address this need, we have developed ProMoST. ProMoST is a freely accessible Web-based application that calculates and displays the mass and pI values for either proteins in the NCBI database identified by accession number or from submitted FASTA format sequence. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223940 Fri, 30 Jan 2009 05:00:00 +0100 2223940 http://www.medworm.com/index.php?rid=2223939&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_20 Over the last 10 years array and mass spectrometry technologies have enabled the determination of the transcriptome and proteome of biological and in particular eukaryotic systems. This information will likely be of significant value to our elucidation of the molecular mechanisms that govern eukaryotic physiology. However, an equally, if not more important goal, is to define those proteins that participate in signalling pathways that ultimately control cell fate. Enzymes that phosphorylate tyrosine, serine, and threonine residues on other proteins play a major role in signalling cascades that determine cell-cycle entry, and survival and differentiation fate in the tissues across the eukaryotic kingdoms. Knowing which signalling pathways are being used in these cells is of critical importan... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223939 Fri, 30 Jan 2009 05:00:00 +0100 2223939 http://www.medworm.com/index.php?rid=2223938&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_2 Differential labelling techniques like differential in-gel electrophoresis (DIGE) enable mixing a control with an experimental sample prior to protein separation, thereby reducing complexity and greatly improving the resolution and analysis of changes in protein expression. Although the shift caused by phosphorylation to a more acidic pI can, in principle, reveal phosphorylation events using DIGE, analysis and verification of the phosphorylation are fraught with problems. Here we describe a differential phospho-labelling technique that obtains the same advantages as DIGE, which we named DIPPL, for differential phosphoprotein labelling. The technique involves labelling two samples, one with 32Pi (orthophosphate) and the other with 33Pi (orthophosphate). The two samples are mixed and protein... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223938 Fri, 30 Jan 2009 05:00:00 +0100 2223938 http://www.medworm.com/index.php?rid=2223937&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_19 In view of the importance of information transfer mediated throughout the cell by recognition, phos-phorylation or dephosphorylation of kinases, their adapters, or substrates, this method was developed. The method provides a potent research tool for rapidly generating and testing these substrates as modeled by synthetic peptide arrays. The peptides or phosphorylated peptides are automatically generated on the inner surfaces of microplate wells, covalently linked to a polylysine polymer so that they are in a sterically favorable conformation, immediately available for in situ testing. Products up to 18 amino acids long have shown excellent mass spectral homogeneity. Thus, determinate peptide libraries can be ready for testing in as little as 2 days after the conception of an experiment. The... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223937 Fri, 30 Jan 2009 05:00:00 +0100 2223937 http://www.medworm.com/index.php?rid=2223936&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_18 Protein tyrosine phosphorylation plays a central role in cell-signaling and is a focus of biomedical studies and cancer therapy. However, it is still challenging to identify or characterize the coordinated changes of many candidate proteins of one particular pathway or multiple pathways simultaneously. Antibody array is a recently developed approach applied for differential analysis of multiple protein posttransla tional modification events in mammalian cells. It is based on the highly specific recognition between the immobilized antibodies on the array and their specific target proteins in a high-throughput screening format. Here we have described in detail two methods for differential analysis of protein tyrosine phos phorylation in cells by (1) using a single fluorescent protein capture... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223936 Fri, 30 Jan 2009 05:00:00 +0100 2223936 http://www.medworm.com/index.php?rid=2223935&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_17 The study of protein phosphorylation in combination with chemical methods may serve several purposes. The removal of the phosphate group from phosphoserine and -threonine residues by β-elimination has been employed to improve sensitivity for mass spectrometric detection and to attach affinity tags for phosphopeptide enrichment. More recently, phosphoramidate chemistry has been shown to be another promising tool for enriching phosphorylated peptides, and other phosphate-directed reactions may also be applicable to the study of the phosphoproteome in the future. In recent years, the combination of large-scale phospho-proteomics studies with stable isotope labeling for quantification purposes has become of growing importance, frequently involving the introduction of chemical tags such as... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223935 Fri, 30 Jan 2009 05:00:00 +0100 2223935 http://www.medworm.com/index.php?rid=2223934&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_16 We present a gel-free proteomics procedure for the specific isolation of phosphorylated peptides from whole proteome digests. Central is the use of diagonal, reverse-phase chromatography which consists of two consecutive reverse-phase peptide separations with a modification step in between. The latter alters the column retention of affected peptides, thereby allowing their specific isolation from the bulk of nonaffected peptides. To isolate phosphopeptides from complex mixtures, this modification step is a dephosphorylation reaction using a cocktail of broad-spectrum phosphatases. Upon dephosphorylation, peptides undergo a hydrophobic shift and are thereby sorted from in vivo nonphosphorylated peptides. To increase the overall yield of phosphopeptides, a pre-enrichment step was found neces... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223934 Fri, 30 Jan 2009 05:00:00 +0100 2223934 http://www.medworm.com/index.php?rid=2223933&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_15 We describe the application of capillary LC-ICP-MS (liquid chromatography–inductively coupled plasma–mass spectrometry) for quantification of phospho-proteins and their phosphorylation degree. Element mass spectrometry is ideally suited for monitor ing and quantification of compounds with heteroelements such as phosphorus and sulphur, particularly because the ICP-MS response is virtually independent from the chemical form of the element. Determina tion of the phosphorylation stoichiometry, i.e. the relative abundance of the phosphorylated isoforms, can be assessed by the relative abundance of phosphorus compared with sulphur as a marker for the protein amount. Moreover, isotope dilution analysis by post-column addition of a 34S-Spike provides absolute protein quantification wit... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223933 Fri, 30 Jan 2009 05:00:00 +0100 2223933 http://www.medworm.com/index.php?rid=2223932&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_14 Electron capture dissociation (ECD) allows fragmentation of the phosphopeptide backbone while keeping the labile phospho-amino acid intact. This feature of ECD fragmentation, coupled with the acquisition of mass spectra at high mass accuracy, makes ECD well-suited to phosphorylation mapping. The following methods are designed to focus ECD events on phosphopeptides within a complex peptide sample, either by using phosphoric acid neutral loss peaks as a trigger or by targeted analysis of predetermined precursor masses. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223932 Fri, 30 Jan 2009 05:00:00 +0100 2223932 http://www.medworm.com/index.php?rid=2223931&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_12 Protein phosphorylation is a reversible and frequently occurring posttranslational modification regulating a large number of biological functions. Understanding the role phosphorylation events play in biochemical pathways requires the detection of phosphorylated proteins and their phosphorylated amino acids. Mass spectrometry has developed as the premier method for characterizing phosphoproteins as it is sensitive to detecting phosphosites within a single protein or a complex protein mixture (phosphoproteomics). Here an overview is provided of the sample separation and mass spectrometry techniques commonly used for qualitative phosphoprotein analysis. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223931 Fri, 30 Jan 2009 05:00:00 +0100 2223931 http://www.medworm.com/index.php?rid=2223930&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_11 Global monitoring of cellular signaling activity is of great importance for the understanding of the regulation of complex signaling networks and the characterization of signaling pathways deregulated in diseases. Tyrosine phosphorylation of intracellular signaling proteins followed by the recognition and binding of Src homology 2 (SH2) domains are key mechanisms in the downstream transmission of many important biological signals. SH2 domains, comprising 120 members in humans, are small modular protein binding domains that recognize tyrosine phosphorylated signaling proteins with high specificity. Based on these binding properties, the large number of naturally occurring and currently available SH2 domains serve as excellent probes for the comprehensive profiling of the cellular state of s... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223930 Fri, 30 Jan 2009 05:00:00 +0100 2223930 http://www.medworm.com/index.php?rid=2223929&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_10 Phospho-proteomics, the global analysis of protein phosphorylation, holds great promise for the discovery of cell signaling events that link changes in dynamics of protein phosphorylation to the progression of various diseases, particularly cancer and diabetes. Mass spectrometry has become a powerful tool for identification and global profiling of protein phosphorylation. However, even with continuously improving sensitivity of mass spectrometers, sub-stoichiometric nature of phosphorylation poses enormous challenges for phosphoprotein identification and, particularly, mapping phosphosites. Therefore, a successful mass spectrometry-based phosphoproteomic experiment depends largely on an effective method of phosphopeptide enrichment. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223929 Fri, 30 Jan 2009 05:00:00 +0100 2223929 http://www.medworm.com/index.php?rid=2223928&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-834-8_1 The two-dimensional (2-D) gel-based proteomics platform remains the workhorse for proteomics and is fueled by a number of key improvements, including fluorescence-based stains for detection and quantification of proteins and phosphoproteins with high sensitivity and linear dynamic ranges. One such stain is Pro-Q diamond phosphoprotein stain (Pro-Q DPS), which binds to the phosphate moiety of phospho-proteins irrespective of the phosphoamino acid. We recently introduced a modified Pro-Q DPS protocol to detect phosphoprotein spots on 2-D gels with very low background addressing some prime concerns, including high cost and reproducibility of Pro-Q DPS. The major modifications were a threefold dilution of Pro-Q DPS and the use of threefold less volume of the diluted staining solution. In this ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223928 Fri, 30 Jan 2009 05:00:00 +0100 2223928 http://www.medworm.com/index.php?rid=2223927&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F%2F978-1-60327-834-8_13 This chapter focuses on the development of new proteomic approaches based on classical biochemical procedures coupled with new mass spectrometry methods to study the phosphorylation, the most important and abundant PTMs in modulating protein activity and propagating signals within cellular pathways and networks. These phosphoproteome studies aim at comprehensive analysis of protein phosphorylation by identification of the phosphoproteins, exact localization of phosphorylated residues, and preferably quantification of the phosphorylation. Because of low stoichiometry, heterogeneity, and low abundance, enrichment of phosphopeptides is an important step of this analysis. The first section is focused on the development of new enrichment methods coupled to mass spectrometry. Thus, improved appr... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=2223927 Fri, 30 Jan 2009 05:00:00 +0100 2223927 http://www.medworm.com/index.php?rid=1948113&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_9 We describe, in detail, an HT-automated purification of hexahistidine-tagged recombinant proteins using MagneHis™ Ni-Particles and the Biomek FX robot. This procedure is universally applicable to hexa-histidine-tagged recombinant proteins with the tag positioned at either the N- or C-terminus. With minor modifications, the automated protein purification protocol presented in this chapter could be adapted to purify recombinant proteins bearing other tags than hexahistidine and/or other expression systems than E. coli. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948113 Tue, 11 Nov 2008 13:05:53 +0100 1948113 http://www.medworm.com/index.php?rid=1948112&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_8 We describe a plate-based cloning and expression strategy for efficient high-throughput generation of validated expression clones in Escherichia coli. The process incorporates 48- or 96-well plates at all stages including the cloning and colony selection phases which are often performed manually. A 48-grid agar growth plate has been integrated into the colony selection component to improve throughput at the cloning stage. The combinations of 48- and 96-well plate formats are compatible with automated liquid handlers and multichannel pipettes. This revised cloning and expression pipeline increases throughput significantly, and also results in a reduction in both time and material requirements. The system has been validated by the production and screening of several thousand clones at the Mi... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948112 Tue, 11 Nov 2008 13:05:53 +0100 1948112 http://www.medworm.com/index.php?rid=1948111&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_7 Fifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate pro tein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948111 Tue, 11 Nov 2008 13:05:53 +0100 1948111 http://www.medworm.com/index.php?rid=1948110&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_6 Significant innovations in molecular biology methods have vastly improved the speed and efficiency of traditional restriction site and ligase-based cloning strategies. “Enzyme-free” methods eliminate the need to incorporate constrained sequences or modify Polymerase Chain Reaction (PCR)-generated DNA fragment ends. The Polymerase Incomplete Primer Extension (PIPE) method further condenses cloning and mutagenesis to a very simple two-step protocol with complete design flexibility not possible using related strategies. With this protocol, all major cloning operations are achieved by transforming competent cells with PCR products immediately following amplification. Normal PCRs generate mixtures of incomplete extension products. Using simple primer design rules and PCR, short, ove... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948110 Tue, 11 Nov 2008 13:05:52 +0100 1948110 http://www.medworm.com/index.php?rid=1948109&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_5 In this chapter, protocols for the construction of expression vectors using In-Fusion™ PCR cloning are presented. The method enables vector and insert DNA sequences to be seamlessly joined in a ligation-independent reaction. This property of the In-Fusion process has been exploited in the design of a suite of multi-host compatible vectors for the expression of proteins with precisely engineered His-tags. Vector preparation, PCR amplification of the sequence to be cloned and the procedure for inserting the PCR product into the vector by In-Fusion™ are described. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948109 Tue, 11 Nov 2008 13:05:52 +0100 1948109 http://www.medworm.com/index.php?rid=1948108&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_4 A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948108 Tue, 11 Nov 2008 13:05:52 +0100 1948108 http://www.medworm.com/index.php?rid=1948107&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_3 The rate-limiting step in protein production is usually the generation of an expression clone that is capable of producing the protein of interest in soluble form at high levels. Although cloning of genes for protein expression has been possible for some time, efficient generation of functional expression clones, particularly for human proteins, remains a serious bottleneck. Often, such proteins are hard to produce in heterologous systems because they fail to express, are expressed as insoluble aggregates, or cannot be purified by standard methods. In many cases, researchers are forced to return to the cloning stages to make a new construct with a different purification tag, or perhaps to express the protein in a different host altogether. This usually requires identifying new cloning sche... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948107 Tue, 11 Nov 2008 13:05:51 +0100 1948107 http://www.medworm.com/index.php?rid=1948106&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_20 We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948106 Tue, 11 Nov 2008 13:05:51 +0100 1948106 http://www.medworm.com/index.php?rid=1948105&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_2 The advent of high-throughput protein production and the vast amount of data it is capable of generat ing has created both new opportunities and problems. Automation and miniaturization allow experimen tation to be performed more efficiently, justifying the cost involved in establishing a high-throughput platform. These changes have also magnified the need for effective statistical methods to identify trends and relationships in the data. The application of quantitative management tools to this process provides the means of ensuring maximum efficiency and productivity. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948105 Tue, 11 Nov 2008 13:05:50 +0100 1948105 http://www.medworm.com/index.php?rid=1948104&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_19 This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. The protease is initially produced as a fusion to the C-terminus of E. coli maltose binding protein (MBP), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. An S2... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948104 Tue, 11 Nov 2008 13:05:50 +0100 1948104 http://www.medworm.com/index.php?rid=1948103&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_18 Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell–cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948103 Tue, 11 Nov 2008 13:05:49 +0100 1948103 http://www.medworm.com/index.php?rid=1948102&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_17 Integral membrane proteins are a major challenge within structural genomics. These proteins are not only difficult to produce in quantities sufficient for analysis by X-ray diffraction or NMR, but also require extraction from their lipid environment, which leads to a new dimension of difficulties in purification and subsequent structural analysis. To overcome these problems requires new strategies enabling screening larger number of parameters dealing with expression and purification. For this reason, we have developed high-throughput methods for screening extracting and purifying detergents as well as other purification parameters, e.g. salt and pH. The method requires standard laboratory equipments, but can also be automated. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948102 Tue, 11 Nov 2008 13:05:49 +0100 1948102 http://www.medworm.com/index.php?rid=1948101&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_16 In this chapter, protocols for the growth and transfection of Human Embryonic Kidney (HEK) 293T cells for small scale expression screening and large scale protein production are described. Transient expression in mammalian cells offers a method of rapidly producing glycoproteins with a relatively high throughput. HEK 293T cells, in particular, can be transfected with high efficiency (> 50% cell expression) and are amenable to culture at multi-litre scale. Growing cells in micro-plate format allows screening of large numbers of vectors in parallel to prioritise those amenable to scale-up and purification for subsequent structural or functional studies. The glycoform of the expressed protein can be modified by treating cell cultures with kifunensine which inhibits glycan processing during... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948101 Tue, 11 Nov 2008 13:05:47 +0100 1948101 http://www.medworm.com/index.php?rid=1948100&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_15 One of the main challenges in this post genomic era is the development and implementation of efficient methods of protein synthesis. A clear understanding of the role of genes in an organism is to comprehend the biological functions of all of its proteins. Acquiring this knowledge will depend in part on the success of rapid synthesis and purification of proteins. The future of structural genomics and functional proteomics depends on the availability of abundantly expressing, soluble proteins in a high-throughput manner. Conventional cell based methods of protein expression is rather laborious, time consuming and the ways to fail are numerous including solubility, toxicity to the host and instability (e.g. proteolysis). Cell-free or in vitro protein synthesis, on the other hand allows the e... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948100 Tue, 11 Nov 2008 13:05:46 +0100 1948100 http://www.medworm.com/index.php?rid=1948099&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_14 The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years hav... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948099 Tue, 11 Nov 2008 13:05:41 +0100 1948099 http://www.medworm.com/index.php?rid=1948098&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_13 We present three complementary approaches for biotinylating proteins in vivo in Escherichia coli or in vitro using chemical or enzymatical reactions all of which can be scaled up to tag large numbers of proteins in parallel. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948098 Tue, 11 Nov 2008 13:05:41 +0100 1948098 http://www.medworm.com/index.php?rid=1948097&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_12 A method has been developed that eliminates the need for complex chromatographic apparatus in the purification of recombinant proteins expressed in Escherichia coli. This method is similar to conventional affinity-tag separations, but the affinity resin is replaced by polyhydroxybutyrate (PHB) particles prodced in vivo in the E. coli expression host during protein expression. A PHB-binding protein known as a phasin is genetically fused to the product protein via an engineered pH and temperature dependent self-cleaving intein linker. Thus the phasin—sion acts as a self-cleaving purification tag, with affinity for the co-expressed PHB granules. The PHB particles and tagged target protein are purified by lysing the cells and washing the granules with sequential rounds of centrifugation ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948097 Tue, 11 Nov 2008 13:05:40 +0100 1948097 http://www.medworm.com/index.php?rid=1948096&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_11 Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His6MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His6MBP fusion protein is cleaved by tobacco etch virus (TEV) protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His6MBP. (Source: Springer protocols feed by Protein Sc... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948096 Tue, 11 Nov 2008 13:05:39 +0100 1948096 http://www.medworm.com/index.php?rid=1948095&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_10 The production of recombinant proteins usually involves the exploration of a wide variety of expression and purification methodologies in the pursuit of a strategy tailored to a particular protein. The methods applied are reliant on exploiting individual differences between expression systems or the variations in specific protein properties. These bespoke strategies have not lent themselves to high-throughput methodologies. Ultimately the development of robust generic methods capable of simplifying and stabilizing the process, allowing automation, was necessary to increase throughput. This chapter describes a series of high-throughput methods used to express, purify, and quantify recombinant protein produced in E. coli or insect cells. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948095 Tue, 11 Nov 2008 13:05:38 +0100 1948095 http://www.medworm.com/index.php?rid=1948094&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-196-3_1 Recombinant protein production plays a crucial role in the drug discovery process, contributing to several key stages of the pathway. These include exploratory research, target validation, high-throughput screening (HTS), selectivity screens, and structural biology studies. Therefore the quick and rapid production of high-quality recombinant proteins is a critical component of the successful development of therapeutic small molecule inhibitors. This chapter will therefore attempt to provide an overview of some of the current “best-in-class” cloning, expression, and purification strategies currently available that enhance protein production capabilities and enable greater throughput. As such the chapter should also enable a reader with limited understanding of the high-throughpu... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1948094 Tue, 11 Nov 2008 13:05:37 +0100 1948094 http://www.medworm.com/index.php?rid=1548320&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_9 The infectious agents of prion diseases are unorthodox, and they seem to be composed primarily of a misfolded glycoprotein called the prion protein (PrP). Replication of prion infectivity is associated with the conversion of PrP from its normal, cellular form (PrPC) into a pathogenic form (PrPSc), which is characterized biochemically by relative detergent insolubility and protease resistance. Several techniques have been developed in which PrPC molecules can be converted into the PrPSc conformation in vitro (1–8). These biochemical techniques recapitulate several specific aspects of in vivo prion propagation (1–3), and one method, the protein misfolding cyclic amplification technique, also has been shown to amplify infectivity (5). In this chapter, we describe a method for ampl... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548320 Wed, 04 Jun 2008 04:00:00 +0100 1548320 http://www.medworm.com/index.php?rid=1548319&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_8 Misfolding and aggregation of prion protein (PrP) is related to several neurodegenerative diseases in humans such as Creutzfeldt—Jacob disease, fatal familial insomnia, and Gerstmann—Straussler—Sheinker disease. Amyloid fibrils prepared from recombinant PrP in vitro share many features of the infectious prions. These fibrils can be used as a synthetic surrogate of PrPSc for development of prion diagnostics, including generation of PrPSc-specific antibody, for screening of antiprion drugs, or for development of antiprion decontamination procedures. Here, we describe the methods of preparation of prion protein fibrils in vitro and biochemical assays for assessing physical properties and the quality of fibrils. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548319 Wed, 04 Jun 2008 04:00:00 +0100 1548319 http://www.medworm.com/index.php?rid=1548318&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_7 A central event in the transmission and pathogenesis of transmissible spongiform encephalopathy diseases is the misfolding of the prion protein. Considerable progress has been made in our understanding of this misfolding event through the development of cell-free assays that mimic the molecular features of prion propagation. This chapter reviews the contribution of cell-free assays to our understanding of prion propagation. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548318 Wed, 04 Jun 2008 04:00:00 +0100 1548318 http://www.medworm.com/index.php?rid=1548317&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_6 Prion peptide (PrP) neurotoxicity has been modelled in vitro by using synthetic peptides derived from the PrPC sequence. The major region of neurotoxicity has been localized to the hydrophobic domain located in the middle of the PrP sequence. The neurotoxicity assays are typically performed on cultured mouse cerebellar neurons derived from neonatal pups, and viability can be monitored by a variety of assays, including MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium); MTS (3-(4,5-dimeth-ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) lactate dehydrogenase release; and apoptotic assays. These neurotoxicity studies have been useful in identifying cofactors, such as PrPC and metals as modulators of PrP peptide-mediated neurotoxicity. Given the... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548317 Wed, 04 Jun 2008 04:00:00 +0100 1548317 http://www.medworm.com/index.php?rid=1548316&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_5 Prion-propagating cell lines are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Use of cell-based models has lead to the finding that prion protein (PrPC) and PrPSc are released from cells in association with exosomes. Furthermore, exosomes have been shown to act as vehicles for infectivity, transferring PrPSc between cell lines and providing a mechanism for prion spread between tissues. As a role for exo-somes in prion disease is emerging, this chapter outlines a method for the generation of prion-infected cell lines and the isolation and characterization of PrPC- and PrPSc-containing exosomes. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548316 Wed, 04 Jun 2008 04:00:00 +0100 1548316 http://www.medworm.com/index.php?rid=1548315&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_4 Prions are usually quantified by bioassays based on intracerebral inoculation of animals, which are slow, imprecise, and costly. We have developed a cell-based prion assay that is based on the isolation of cell lines highly susceptible to certain strains (Rocky Mountain Laboratory and 22L) of mouse prions and a method for identifying individual, prion-infected cells and quantifying them. In the standard scrapie cell assay (SSCA), susceptible cells are exposed to prion-containing samples for 4 days, grown to confluence, passaged two or three times, and the proportion of rPrPSc-containing cells is determined with automated counting equipment. The dose response is dynamic over 2 logs of prion concentrations. The SSCA has a standard error of ±20–30%, is as sensitive as the mouse b... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548315 Wed, 04 Jun 2008 04:00:00 +0100 1548315 http://www.medworm.com/index.php?rid=1548314&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_3 Prion infectivity is often linked to presence of the protease-resistant isoform of prion protein (PrP), PrPres; therefore, it is of highest interest to have convenient methods for rapid detection of PrPres in the research laboratory. For detection of PrPres in model systems to confirm infectivity, there are several methods that can be applied. This chapter focuses on detection of PrPres by proteinase K digestion followed by Western blot, which is the only method that is both quantitative and qualitative. For large-scale screening of PrPres content in samples, the dot blot method offers a great advantage for detecting PrPres, and this method is also thoroughly described in this chapter. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548314 Wed, 04 Jun 2008 04:00:00 +0100 1548314 http://www.medworm.com/index.php?rid=1548313&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_2 Prion protein (PrP)C expression levels and protein localization are known to be affected by factors such as metal ions and oxidative stress. By the development of a green fluorescent protein (GFP)-PrPC fusion protein, the movement of PrP can be followed in real time. Furthermore, alterations in cellular metabolism can be detected while cells are still viable. The internalization response of PrP to 20 μM manganese (Mn) in divalent metal ion-depleted media is used to demonstrate the movement of GFP-tagged proteins in live cells and real tim0e. A live cell microtiter plate assay shows that PrP null cells are less capable of dealing with Mn-induced oxidative stress. In addition, this chapter outlines several complementary techniques for studying live cells and GFP fusion proteins. (Source: ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548313 Wed, 04 Jun 2008 04:00:00 +0100 1548313 http://www.medworm.com/index.php?rid=1548312&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_18 The unconventional nature of the infectious agent of prion diseases poses a challenge to conventional infection control methodologies. The extra neural tissue distribution of variant and sporadic Creutzfeldt—Jakob disease has increased concern regarding the risk of prion disease transmission via general surgical procedures and highlighted the need for decontamination procedures that can be incorporated into routine processing. This chapter describe a quantitative method for assessing the prionocidal activity of chemical and physical decontamination methods against surface-bound prion infectivity. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548312 Wed, 04 Jun 2008 04:00:00 +0100 1548312 http://www.medworm.com/index.php?rid=1548311&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_17 Prions represent a new biological paradigm of protein-mediated information transfer. In mammals, prions are the cause of fatal, transmissible neurodegenerative diseases, often referred to as transmissible spongiform encephalopathies. Many unresolved issues remain, including the exact molecular nature of the prion, the detailed mechanism of prion propagation, and the mechanism by which prion diseases can be both genetic and infectious. In addition, we know little about the mechanism by which neurons degenerate during prion diseases. Tied to this, the physiological function of the normal form of the prion protein remains unclear, and it is uncertain whether loss of this function contributes to prion pathogenesis. The factors governing the transmission of prions between species remain unclear... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548311 Wed, 04 Jun 2008 04:00:00 +0100 1548311 http://www.medworm.com/index.php?rid=1548310&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_16 Within the spectrum of sporadic human transmissible spongiform encephalopathies (TSEs), there is considerable diversity of disease phenotypes. At least part of this variation is thought to be on the basis of different “strains” of prions (the infectious agent). Tissue deposition of PrPres (the abnormal disease-associated conformation of the prion protein) is considered a hallmark of TSE pathology, and it can be visualized by Western blotting typically as three bands depicting the diglycosylated, monoglycosylated, and unglycosylated species. It is the mobility of the unglycosylated PrPres, and the relative abundance of the two glycosylated bands, along with the prion protein gene (PRNP) codon 129 genotype, that seem to correlate with distinct clinico-pathological profiles of spo... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548310 Wed, 04 Jun 2008 04:00:00 +0100 1548310 http://www.medworm.com/index.php?rid=1548309&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_15 Numerous transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins, covering a vast range of structural and functional classes, are recognized to undergo proteolytic cleavage or shedding from the plasma membrane. Although this widespread phenomenon seems fundamental to normal cellular biology, proteolytic processing also seems to play a central role in the pathogenesis of some neurodegenerative disorders such as Alzheimer's disease. An analogous situation may exist in prion disorders. The GPI-anchored cellular prion protein (PrPC) may be endoproteolytically cleaved at two different sites: one at the C-terminal end of the octameric repeat region and the other within a potentially neurotoxic and amyloidogenic region of the protein. The relevance of these alternative proteolytic ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548309 Wed, 04 Jun 2008 04:00:00 +0100 1548309 http://www.medworm.com/index.php?rid=1548308&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_14 Human prion diseases are associated with a range of clinical presentations, and they are classified by both clinicopathological syndrome and etiology, with subclassification according to molecular criteria. Here, we describe procedures that are used within the MRC Prion Unit to determine a molecular diagnosis of human prion disease. Sequencing of the PRNP open reading frame to establish the presence of pathogenic mutations is described, together with detailed methods for immunoblot or immunohistochemical determination of the presence of abnormal prion protein in brain or peripheral tissues. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548308 Wed, 04 Jun 2008 04:00:00 +0100 1548308 http://www.medworm.com/index.php?rid=1548307&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_13 The binding of paramagnetic metal ions is thought to be an essential function of the prion protein and lends itself to interrogation by electron paramagnetic resonance (EPR), which probes the local coordination environment of bound metal ions to provide details of the metal-binding affinity, stoichiometry, and the symmetry and identity of its ligating atoms. It is also capable of identifying reactive oxygen/nitrogen species and peptide-derived radicals, in addition to monitoring protein-membrane dynamics and conformation by using site-directed spin labeling. An overview of the EPR technique as applied to the prion protein is given, key results are summarized, and some future experimental avenues are outlined. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548307 Wed, 04 Jun 2008 04:00:00 +0100 1548307 http://www.medworm.com/index.php?rid=1548306&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_12 The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to be a metallo-protein that binds copper (Cu), and copper might have a role in the normal function of the protein. Conversely, PrP expression in yeast led us to suggest that the protein might be involved in the regulation of Cu homeostasis. In the presence of excess Cu in the growth medium, PrP expression limited the increase of the total number of Cu atoms per cell to a maximum of 14-fold compared with mock control cells, which showed a 52-fold increased intracellular Cu level. Conclusively, we suggest that PrP expression itself has a regulatory or buffering function for the cellular Cu level in yeast cells, most likely due to binding of Cu to the multi... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548306 Wed, 04 Jun 2008 04:00:00 +0100 1548306 http://www.medworm.com/index.php?rid=1548305&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_11 The availability of recombinant prion proteins (recPrP) has been exploited as a model system to study PrP-mediated toxicity, conversion, and infectivity. According to the protein only hypothesis, the central event in the pathogenesis of prion diseases is the conversion of PrPC to PrPSc. This involves a dramatic increase in β sheet conformation as PrPC is converted to PrPSc, and it is widely believed that this conformational change affects the as-yet undefined function of PrPC. Although there are many methods available to monitor for the changes in the structural makeup of PrP mutants and oligomers formed with respect to disease relevance, circular dichroism is one of the most popular methods used. In this chapter, we discuss the fundamental principles of circular dichroism and its cur... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548305 Wed, 04 Jun 2008 04:00:00 +0100 1548305 http://www.medworm.com/index.php?rid=1548304&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_10 Certain applications in the prion field require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, which are normally generated as a result of spontaneous oxidation or degradation. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548304 Wed, 04 Jun 2008 04:00:00 +0100 1548304 http://www.medworm.com/index.php?rid=1548303&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-234-2_1 From an early stage of prion research, tissue cultures that could support and propagate the scrapie agent were sought after. The earliest attempts were explants from brains of infected mice, and their growth and morphological characteristics were compared with those from uninfected mice (1). Using the explant technique, several investigators reported increased cell growth in cultures established from scrapie-sick brain compared with cultures from normal mice (1, 2). These are odd findings in the light of the massive neuronal cell death known to occur in scrapie-infected brains; however, the cell types responsible for the increased cell growth in the scrapie-explants most probably were not neuronal. The first successful cell culture established in this way, in which the scrapie agent was se... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1548303 Wed, 04 Jun 2008 04:00:00 +0100 1548303 http://www.medworm.com/index.php?rid=1539213&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_23 One of the most important structural features of recombinant monoclonal antibodies produced in mammalian cells is the N-linked oligosaccharide profile. These profiles impact recombinant therapeutics in a multitude of ways affecting distribution, efficacy, and immunogenicity. High mannose, α-gal and other oligosaccharide species are highly immunogenic and in most cases should be minimized during manufacturing. A recombinant monoclonal antibody, h5G1.1, was produced in NS0 and CHO cell lines and tested to identify changes in the N-linked oligosaccharide profiles caused from a change in cell line. Traditional peak analysis using HPLC with fluorescence detection was augmented by mass spectrometric analysis. Nano LC-MS following tryptic digestion corroborated HPLC findings of the presence... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539213 Fri, 04 Apr 2008 04:00:00 +0100 1539213 http://www.medworm.com/index.php?rid=1539212&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_13 Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-P-Manα1–2Manα1–6Manα1–4GlcNH2) linked to the 6-position of the D-myo-inositol ring of phos-phatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. The preassembled GPI-anchor precursor is post-translationally transferred to a variety of membrane proteins in the lumen ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539212 Fri, 04 Apr 2008 04:00:00 +0100 1539212 http://www.medworm.com/index.php?rid=1539211&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_22 Majority of protein drugs in development today are glycoproteins e.g. recombinant antibodies expressed in various cell lines. Oligosaccharides through conformational changes can modulate therapeutic value (potency) of glycoproteins e.g. complement dependent cell cytotoxicity (CDCC) and antibody-dependent cell cytotoxicity (ADCC) activities of MAbs. Carbohydrate structure analysis in detail is an integral part of protein drug characterization. This not only allows understanding of carbohydrates, but may allow deeper insight into the structure-function of the whole protein molecule. Oligosaccharide mapping by HPLC with fluorescence detection is a powerful technique that sheds considerable light into understanding of glycan structures with minimal effort. Oligosaccharide analysis using pulsed... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539211 Fri, 04 Apr 2008 04:00:00 +0100 1539211 http://www.medworm.com/index.php?rid=1539210&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_20 Secreted as well as membrane-associated eukaryotic proteins are most commonly glycosylated. Saccharides are attached to proteins mainly through N-and O-glycosydic bonds or as part of the glycosylphosphatidyinositol-membrane anchor. In contrast to N-glycosylation, which involves the co-translational transfer in the endoplasmic reticulum (ER) of the glycan portion of Glc3Man9GlcNAc2-PP-dolichol to suitable Asn residues on nascent polypeptides, O-glycosylation begins with the addition of a single monosaccharide. Contrary to N-glycosylation, which involves an asparagine residue in the sequon Asn-Xaa-Thr/Ser (Xaa can be any amino acid except Pro, and it is rarely Cys), no particular sequence motif has been described for O-glycosylation. This may reflect the fact that: (1) the specificity of the... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539210 Fri, 04 Apr 2008 04:00:00 +0100 1539210 http://www.medworm.com/index.php?rid=1539209&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_18 Enzymatic sequencing of oligosaccharides gives structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can be applied to oligosaccharide mixtures as well to evaluate the occurrence of distinct oligosac-charide motives of functional and/or structural interest. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539209 Fri, 04 Apr 2008 04:00:00 +0100 1539209 http://www.medworm.com/index.php?rid=1539208&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_16 When performing a structural analysis of N-glycans, a number of aspects should be considered. N-Glycans may be hydrolyzed from purifi ed glyc-oproteins, serum glycoprotein mixtures, or delipidated membrane fractions by chemical hydrolysis using hydrazine or enzymatic hydrolysis using PNGase F. Chemical deglycosylation may be an economical alternative to produce N-and O-glycans in a preparative scale, but it is less suitable for analytical purposes. By chemical hydrazinolysis the protein core is destroyed completely and all acyl groups are cleaved from neuraminic acid residues as well as from N-acetylhexosamine residues. If not only a structure analysis of N-glycans is intended but a sequencing of the protein core, an analysis of the different types of neuraminic acids or an elucidation of ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539208 Fri, 04 Apr 2008 04:00:00 +0100 1539208 http://www.medworm.com/index.php?rid=1539207&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_2 Post-translational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial in the discovery of biomarkers of disease. Recent commercial availability of fluorescent dyes specifically staining PTMs of proteins such as phosphorylation and glycosylation enables the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular and signalling interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as Multiple Sclerosis (MS), using its animal model, Experimental autoimmune encephalomyelitis (EAE), we have applied the phopshorylation specific fluores... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539207 Fri, 04 Apr 2008 04:00:00 +0100 1539207 http://www.medworm.com/index.php?rid=1539206&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_5 α-Amidation is a terminal modification in peptide biosynthesis that can itself be rate-limiting in the overall production of bioactive α-amidated peptides. More than half of the known neural and endocrine peptides are α-amidated and in most cases, this structural feature is essential for receptor recognition, signal transduction, and thus, biologic function. This chapter describes methods for developing and using analytical tools to study the biology of α-amidated peptides. The principle analytical method used to quantify α-amidated peptides is the radioimmunoassay (RIA). Detailed protocols are provided for 1) primary antibody production and characterization; 2) radiolabeling of RIA peptides; 3) sample preparation; and 4) the performance of the RIA itself. Tec... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539206 Fri, 04 Apr 2008 04:00:00 +0100 1539206 http://www.medworm.com/index.php?rid=1539205&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_1 Oxidation of sulfhydryl groups to form a disulfi de bond is one of the most common post-translational modifi cations in proteins. Disulfi de bonds play important roles in stabilizing three-dimensional structure and modulating bioactivity of the cystinyl proteins. The determination of disulfi de bond linkage is therefore an integral part of structural characterization of proteins. A mass spectrometry-based strategy utilizing chemical cleavage at cysteine residues following cyanylation reaction is described for the identifi cation of both sulfhydryl and disulfi de bond linkage in proteins. The method has been particularly powerful for the assignment of disulfi de bonds in proteins containing adjacent or closely spaced cysteines. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539205 Fri, 04 Apr 2008 04:00:00 +0100 1539205 http://www.medworm.com/index.php?rid=1539204&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_17 Most glycoproteins carry a very heterogeneous mixture of oligosaccharides and even a single glycosylation site of a pure glycoprotein is often heterogeneously glycosylated. The structural diversity of oligosaccharides arises from linkage variants, from differences in the size and number of charges of glycans, and from differences in the monosaccharide composition of glycans. Fortunately, the biosynthetic pathway is subject to certain restrictions, so that structural diversity is limited and amenable to laboratory investigation. Different approaches have been developed to the structural characterization of oligosaccharides, including nuclear magnetic resonance (NMR), mass-spectrometry, linkage analysis by gas chromatography-mass spectrometry (GC-MS), sequence analysis using specific exoglyc... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539204 Fri, 04 Apr 2008 04:00:00 +0100 1539204 http://www.medworm.com/index.php?rid=1539203&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_25 The increase in the number of Web-based resources on post-translational modification sites (PTMSs) in proteins is accelerating. The paper presents a set of computational protocols describing how to work with the Internet resources when dealing with PTMSs. The protocols are intended for querying in PTMSs related data bases, search of the PTMSs in the protein sequences and structures, calculating the pI and molecular mass of the PTM isoforms. Thus, the modern bioinformatics prediction tools make feasible to express protein modification in broader quantitative terms. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539203 Fri, 04 Apr 2008 04:00:00 +0100 1539203 http://www.medworm.com/index.php?rid=1539202&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_8 Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. The ubiquitinated peptide thus has two N-termini – one from the original peptide and one from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain. Any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the “normal” peptide will vary ... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539202 Fri, 04 Apr 2008 04:00:00 +0100 1539202 http://www.medworm.com/index.php?rid=1539201&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_15 Oligosaccharides in glycoproteins by their very nature infl uence many aspects of protein function, e.g., half-life and activity/potency. Recombinant IgGs constitute a major portion of therapeutic proteins. Though the glycans in IgGs account for about 2% of the total weight, they influence biologic activity apart from antigen binding. Characterization of the carbohydrates is not only a regulatory requirement but it may allow understanding of structure-function of proteins. Current advances in analytical techniques permit structural elucidation of small quantities of glycoproteins. At a fi rst glance monosaccharide analysis may provide insight into the types of glycosylation similar to information afforded by amino acid composition. It is the only stand-alone technique by which individual s... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539201 Fri, 04 Apr 2008 04:00:00 +0100 1539201 http://www.medworm.com/index.php?rid=1539200&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_19 for the rapid screening of specific post-translational modifications antibody-based methods are very well suited and applicable without demanding expenditure. Here we describe the immunochemical detection of the O-glycosidically linked cytosolic N-acetylglucosamine modification of proteins, which has attracted increasing interest in the last years. Two different monoclonal antibodies were used in enzyme-linked immunosorbent assays (ELISA), Western blots of 1- and 2- dimension (1D and 2D) separated proteins and immunohistochemical analysis of tissue sections. Slight differences in the recognition of this post-translational epitope by the 2 antibodies are observed and will be discussed. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539200 Fri, 04 Apr 2008 04:00:00 +0100 1539200 http://www.medworm.com/index.php?rid=1539199&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_12 Palmitoylation or S-acylation is the post-translational attachment of fatty acids to cysteine residues and is common among integral and peripheral mem brane proteins. Palmitoylated proteins have been found in every eukaryotic cell type examined (yeast, insect, and vertebrate cells), as well as in viruses grown in these cells. The exact functions of protein palmitoylation are not well understood. Intrin sically hydrophilic proteins, especially signaling molecules, are anchored by long chain fatty acids to the cytoplasmic face of the plasma membrane. Palmitoylation may also promote targeting to membrane subdomains enriched in glycosphingolip ids and cholesterol or affect protein–protein interactions. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539199 Fri, 04 Apr 2008 04:00:00 +0100 1539199 http://www.medworm.com/index.php?rid=1539198&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_3 Phosphorylation, the process by which a phosphate group is attached to a pre-existing protein, is an evolutionarily and metabolically cheap way to change the protein's surface and properties. It is presumably for that reason that it is the most wide-spread protein modification: an estimated 10–30% of all proteins are subject to phosphorylation. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539198 Fri, 04 Apr 2008 04:00:00 +0100 1539198 http://www.medworm.com/index.php?rid=1539197&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_10 ISG15 is a ubiquitin-like modifi er that is conjugated to target proteins by a sequential reaction catalyzed by E1/E2/E3 enzymes (protein ISGylation). ISG15 and protein ISGylation are upregulated by interferon stimuli. ISG15 functions as an antiviral protein against Sindbis virus and HIV-1, but the molecular mechanism remains unknown. Here we describe in detail methods for detecting and analyzing protein ISGylation. The methods consist of plasmid transfection and affi nity purifi cation of ISGylated proteins. In addition, we describe a method for detecting ISGylation of a target protein, Ubc13. (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539197 Fri, 04 Apr 2008 04:00:00 +0100 1539197 http://www.medworm.com/index.php?rid=1539196&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_7 Collagens represent a large family of structurally related extracellular matrix proteins containing unique triple helical structure. One of the characteristics of this structural protein is its extensive post-translational modifications that have major effects on molecular assembly, stability, and metabolism. Hydroxylation of specific lysine residues is one of such unique modifications found in collagen, and the pattern/extent of this modification influences fibrillogenesis, cross-linking, and matrix mineralization. The formation of covalent intermolecular cross-linking is the final modification in collagen biosynthesis and is critical for the stability of collagen. The process of cross-linking is dynamic and the pathways vary depending on the tissues and tissue's physiological state. This... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539196 Fri, 04 Apr 2008 04:00:00 +0100 1539196 http://www.medworm.com/index.php?rid=1539195&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_21 Even if a consensus sequence has been identified for a post- translational modification, the presence of such a sequence motif only indicates the possibility, not the certainty that the modification actually occurs. Proteins can be glycosylated on certain amino acid side-chains, and these modifications are designated as N- and O-glycosylation. N-glycosylated species are modified at Asn residues. There is a consensus sequence for N-glycosylation: AsnXxxSer/Thr/Cys, where Xxx can be any amino acid except proline. N-linked oligosaccharides share a common core structure of GlcNAc2Man3. In addition, an enzyme, peptide N-glycosidase F (PNGase F), removes unaltered most of the common N-linked carbohydrates from proteins while hydrolyzing the originally glycosylated Asn residue to Asp. O- glycosyl... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539195 Fri, 04 Apr 2008 04:00:00 +0100 1539195 http://www.medworm.com/index.php?rid=1539194&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_9 Reversible attachment of SUMO (small ubiquitin related modifi er) regulates a large number of proteins and plays an important role in processes such as transcriptional regulation, nucleo-cytoplasmic transport, genome integrity, and cell cycle progression. The steady state level of most sumoylated proteins is very low, presumably caused by strictly regulated modifi cation and/or rapid cycles of modifi cation and de-modifi cation. This often causes a detection problem of sumoylation in vivo. One approach to overcome this obstacle is described here and involves enrich ment of sumoylated proteins under denaturing conditions. After sumoylation is veri fied, addressing its functional consequences is the logical next step. This will benefit signifi cantly from the availability of large quantities... Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539194 Fri, 04 Apr 2008 04:00:00 +0100 1539194 http://www.medworm.com/index.php?rid=1539193&cid=s_37131_60_f&fid=37131&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-084-7_4 Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown to be a common modification and since then an increasing number of proteins have been identified as sulfated. The target proteins belong to the classes of secretory, plasma membrane, and lysosomal proteins, which reflects the intracellular localization of the enzymes catalyzing Tyr sulfation, the tyrosylprotein sulfotransferases (TPSTs). (Source: Springer protocols feed by Protein Science) Springer protocols feed by Protein Science info http://www.medworm.com/rss/comments.php?id=1539193 Fri, 04 Apr 2008 04:00:00 +0100 1539193