http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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-chromatography columns are described. Detailed procedures to mount, test, and operate the system are outlined, and advices for an effective troubleshooting are provided. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 fashion to mass spectrometric detection. This method offers the possibility to analyze high-complex peptide mixtures in a single experiment. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 ions is selected for subsequent MS/MS analysis. Although protein hydrophobicity and size play minor roles (as long as protease cleavage sites are accessible), low copy number can severely limit identification rates. To reduce the amount of peptides entering the mass spectrometer simultaneously without the loss of overall proteomic information, different techniques have been developed. Among these, an approach is represented by COFRADIC (Combined Fractional Diagonal Chromatography). 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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, (3) in-gel digestion and extraction of peptides, (4) labeling of peptides, (5) pooling the samples to be compared, (6) LC-MS/MS of labeled peptides, and (7) database search. The presented workflow is well suited for protein samples of moderate complexity (i.e., protein samples of 300–400 components), and it is exemplified by using different amounts of 25S [U4/U6.U5] tri-snRNPs. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 describe the protocol of redox proteomics, a tool to identify post-translational modifications of proteins. We focus our attention on the identification of carbonylated and 4-hydroxy-2-trans-nonenal-bound proteins. In redox proteomics, samples for the identification of protein carbonyls are first derivatized with 2,4-dinitrophenolhydrazine (DNPH) followed by two-dimensional (2D) separation of these proteins based on their isoelectric point and rate of migration. The carbonylated proteins are then detected using 2D Western blot techniques. Similarly, HNE-bound proteins can be detected using the above-mentioned strategy except that the sample does not need to be derivatized. Separated proteins are identified following tryptic digestion, mass spectrometry, and interrogation of appropriate databases. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 chromatography–tandem mass spectrometry (LC/MS/MS). The first method describes an immobilized metal affinity chromatography (IMAC) technique using Ga3+ to enrich for phosphopeptides from protein digests. The second method describes the utilization of phosphoprotein isotope-coded solid-phase tags (PhIST) to label and enrich phosphopeptides from complex mixtures to both identify and quantify changes in protein phosphorylation. The IMAC and PhIST protocols can be applied to any isolated protein sample and is amenable to additional fractionation using strong cation/anion exchange chromatography prior to reversed-phase LC/MS/MS analysis. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 identification of only one class of membrane proteins. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 protects protein structure and function. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 analysis using strong cation exchange chromatography (SCXC) and liquid chromatography–tandem mass spectrometry (LC/MS/MS). In our approach, microsomes are isolated from plant tissue and then subjected to a two-phase extraction procedure to enrich for plasma membranes. Proteins are extracted and solubilized from the membrane using a methanol-aqueous buffer system that allows for effective reduction, cysteinyl alkylation, and tryptic digestion for subsequent SCXC–LC/MS/MS analysis. Our protocol is also amenable to isotope labeling methods to quantify integral membrane protein expression and post-translational modifications. In addition to plants, the method can be applied to other systems quite readily; thus, we anticipate that it will be of general interest to those characterizing plasma membrane proteins of any organism. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the λ-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463–468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531–537, 2005). 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 clinical proteomics allowing the reproducible profiling of urine. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 basic MALDI sample preparation steps and two easy-to-follow examples for protein identification including extensive notes on these topics with practical tips that are often not available in the Subheadings 2 and 3 of research articles. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 identified. In addition to subunits β1 and β2, the corresponding inducible subunits being part of the immuno-proteasome were identified. The subunit β5i was found to completely replace the corresponding constitutive subunit, suggesting a high proteolytic activity of the intestinal proteasome leading to increased production of antigenic peptides. The high mass accuracy in the low ppm range and resolution of FTICR-MS provide direct identifications of individual proteins as mixtures such as components resulting from incomplete electrophoretic separation. In addition, the comparison of UV- and IR-MALDI FTICR-MS may provide details of fragmentation and rearrangement reactions that may occur under UV-MALDI ionisation conditions. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 minimize methodical variations in spot positions and in protein abundance, to simplify image analysis, as well as to improve protein quantitation by including an internal standard.The most important properties of protein detection methods applied in proteome analysis include high sensitivity (i.e., low detection limit), wide linear dynamic range for quantitative accuracy, reproducibility, cost-efficiency, ease of use, and compatibility with downstream protein identification or characterization technologies, such as mass spectrometry (MS). Regrettably, no single detection method meets all these requirements, albeit fluorescence-based technologies are currently favored for most applications; hence, the major focus of this chapter is on fluorescent-dye-based protein detection and quantitation techniques. Although satisfying results with respect to sensitivity and reproducibility are also obtained by methods based on radioactive labeling of proteins (which is still unsurpassed in terms of sensitivity), radiolabeling is, however, largely impractical for routine proteomic profiling because of the costs and the health and safety concerns associated with handling radioactive compounds. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 electrophoretic techniques can substitute for chromatographic isolation of protein complexes, and can even isolate supramolecular physiological assemblies. Subsequent resolution in second dimension can be denaturing to resolve the subunits of complexes, as exemplified with BNE/SDS 2-DE, or native like in BNE/BNE 2-DE (the latter using different cathode buffers for 1-D BNE and 2-D BNE). After isolation of highly pure membrane protein complexes by two native electrophoretic separations, the separation protocol may be finished by denaturing 2-DE like BAC/SDS or doubled SDS-PAGE. Thus, a four-dimensional electrophoretic system with minimal loss of protein results that is useful as an efficient micro-scale protein separation protocol, e.g. for mass spectrometric analyses. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-157-8_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 with IPGs has largely overcome the former limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling, resolution, and separation of very acidic or basic proteins. Current research to further advance 2-DE technology has focused on improved solubilization/separation of hydrophobic proteins, display of low abundance proteins, and reliable protein quantitation by fluorescent dye technologies. Here, we provide a comprehensive protocol of the current high-resolution 2-DE technology with IPGs for proteome analysis and describe in detail the individual steps of this technique, i.e., sample preparation and protein solubilization, isoelectric focusing in IPG strips, IPG strip equilibration, and casting and running of multiple SDS gels. Last but not the least, a section on how to circumvent the major pitfalls is included. 2009-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-394-7_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) software-supported read-out and data analysis. We review some general concepts about peptide arrays on planar supports and point out technical aspects concerning the generation of peptide microarrays. Finally, we discuss recent applications by describing relevant literature. 2009-08-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-394-7_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. 2009-08-01T04:00:00Z