http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_1 The term “peptidomics” can be defined as the systematic analysis of the peptide content within a cell, organelle, tissue or organism. The science of peptidomics usually refers to the studies of naturally occurring peptides. Another meaning refers to the peptidomics approach to protein analysis. An ancient Roman strategy divide et impera (divide and conquer) reflects the essence of peptidomics. Most effort in this field is spent purifying and dividing the peptidomes, which consist of tens, hundreds or sometimes thousands of functional peptides, followed by their structural and functional characterisation. This chapter introduces the concept of peptidomics, outlines the range of methodologies employed and describes key targets – the peptide groups which are often sought after in such studies. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_10 Direct MALDI-TOF mass spectrometric peptide profiling is increasingly used to analyze the peptide complement in the nervous system of a variety of invertebrate animals from leech to Aplysia and many arthropod species, especially insects and crustaceans. Here, we describe a protocol for direct peptide profiling of defined areas of the central nervous system of insects. With this method, one can routinely and reliably obtain neuropeptide signatures of selected brain areas from various insects. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_11 Today, commercially available mass spectrometers increasingly meet all the demands of the proteomics community including high throughput, high sensitivity, and significant fragmentation capability for sequence determinations. Therefore, proper sample preparation is often the most crucial step to obtain the necessary data, particularly when working with biological samples. Depending on the size, sample preparation techniques differ and have to be optimized empirically. This is particularly apparent at the single cell level. In this chapter, we describe protocols for the use of MALDI-TOF mass spectrometry to directly analyse the peptidome of single insect neurons. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_12 Skin secretions from anurans (frogs and toads), particularly those species belonging to the Hylidae and Ranidae families, are a rich source of biologically active peptides. Cytolytic peptides with broad-spectrum antimicrobial activities and highly variable amino acid sequences are often released into these secretions in high concentrations. Identification and characterization of these components can prove to be valuable in species identification, elucidation of evolutionary histories and phylogenetic relationships between species, and may lead to development of agents with potential for therapeutic application. This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release in a procedure that does not appear to cause distress to the animals. The peptide components in the secretions are separated by reversed-phase HPLC on octadecylsilyl silica (C18) columns under standard conditions after partial purification on Sep-Pak cartridges. Individual peptides are identified by determination of their molecular masses by MALDI-TOF mass spectrometry and from their retention times. The use of mixtures of synthetic peptides of appropriate molecular mass as calibration standards enables mass determination to a high degree of precision. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_13 Antimicrobial peptides (AMPs) play an important role in the host’s innate defence system in many organisms. Amphibian skin is expected to be a particularly rich source of novel AMPs. In amphibians, AMPs are produced from precursor proteins via specific cleavage by processing enzymes. While the nucleotide sequences of the AMP coding region in precursors are hypervariable, those of other regions, including the 5′- and 3′-untranslated regions (UTRs), are highly or relatively conserved in different precursors. Such nucleotide sequence conservation suggests an efficient strategy for molecular cloning of the antimicrobial peptide genes by 3′-rapid amplification of cDNA ends (3′-RACE) and reverse transcriptase polymerase chain reaction (RT-PCR) methods using specific primers. With this strategy in mind we have established an efficient protocol suitable for amplification of multiple cDNAs encoding amphibian AMP precursor proteins. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_14 A large number of diverse antimicrobial peptides have been found in amphibian skins, and many more remain to be identified. It is sufficiently easy to obtain amounts of gland secretions sufficient for both identification and functional testing of the bioactive peptides. We describe here a systematic peptidomics approach which we combined with genomics and functional testing. This has proven to be an effective way to identify amphibian antimicrobial peptides, including novel peptide families. Protocols are exemplified for Bombina maxima and Odorrana grahami and can be easily adapted for use with other amphibian species. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_15 Endocrine tissues like the pituitary, hypothalamus and islets of Langerhans are rich in bioactive peptides. These are used for intercellular signalling and are involved in regulation of almost all physiological processes. Peptidomics is the comprehensive analysis of peptides in tissues, fluids and cells. Peptidomics applied to (neuro-)endocrine tissues aims therefore to identify as many bioactive peptides as possible. Peptidomics of (neuro-)endocrine tissues requires an integrated approach that consists of careful sample handling, peptide separation techniques, mass spectrometry and bioinformatics. Here we describe the methods for isolation and dissection of endocrine tissues, the extraction of bioactive peptides and further sample handling and identification of peptides by mass spectrometry and hyphenated techniques. We also present a straightforward method for the comparison of relative levels of bioactive peptides in these endocrine tissues under varying physiological conditions. The latter helps to elucidate functions of the bioactive peptides. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_16 Here we report our approach to the peptidomic analysis of mouse liver. We use ultrafiltration for peptide prefractionation, which is followed by size exclusion chromatography. The low molecular weight peptides (MW below ~3 kDa) are analysed directly by nanoLC-MS/MS, and the higher molecular weight peptides (MW above ~3 kDa) are characterized with MALDI-TOF MS first and then proteolytically digested prior to nanoLC-MS/MS analyses. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_17 Due to the complexity of the mammalian central nervous system, neuropeptidomic studies in mammals often yield very complicated mass spectra that make data analysis difficult. Careful sample preparation and extraction protocols must be employed in order to minimize spectral complexity and enable extraction of useful information on neuropeptides from a given sample. Controlling post-mortem protease activity is essential to simplifying mass spectra and to identifying low-abundance neuropeptides in tissue samples. Post-mortem microwave-irradiation coupled with cryostat dissection has proven to be effective in arresting protease activity to allow detection of endogenous neuropeptides instead of protein degradation products. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_18 An emerging way to study neuropsychiatric or neurodegenerative diseases is by performing proteomic analyses of brain tissues. Here, we describe methods used to isolate and identify the proteins associated with a sample of interest, such as the synapse, as well as to compare the levels of proteins in the sample under different conditions. These techniques, involving subcellular fractionation and modern quantitative proteomics using isotopic labels, can be used to understand the organization of neuronal compartments and the regulation of synaptic function under various conditions. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_19 A key challenge in clinics is the identification of sensitive and specific biomarkers for early detection, prognostic evaluation, and surveillance of disease. A biomarker is defined as a biological substance that can be used to specifically detect a disease, measure its progression, or the effect of a treatment. A biomarker should be easily accessible, and ideally sensitivity and specificity must be sufficient to distinguish between false positives, false negatives, and true positives. To be useful for routine clinical evaluation, a biomarker should be detectable in body fluids (e.g., plasma, serum, urine). A biomarker can be a metabolite, a specific post-translational modification, a lipid, a phospholipid, or a protein. Due to technical advances in the analysis of biomolecules by mass spectrometry (MS), investigations of peptide biomarkers have increased. In contrast to genome, the peptidome is dynamic and constantly changing. Elucidating how the peptides complement changes in a cell type in diseases is crucial to understand how these processes occur at a molecular level. Lymphoblastoid cell lines, derived from blood lymphocytes, represent suitable models for biochemical investigations and biomedical applications because of their stability, the ease of amplification, and long-term preservation. Technological improvements of MS and liquid chromatography (LC) during the last 10 years resulted in the development of highly sensitive approaches for proteomic and peptidomic analyses. Here we provide guidelines for the preparation of the lymphoblastoid cell lines, the extraction of the peptides and their purification. We describe a number of technologies which we developed for the peptidomic profiling of lymphoblastoid cell extracts from patients with leukodystrophies, linked to mutations in the genes encoding the eukaryotic initiation factor 2B (eIF2B; eIF2B-related disorders). 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_20 Recent years have seen great advances in mass spectrometry and proteomics, the science dealing with the analysis of proteins, their structure and function. A branch of proteomics dealing with naturally occurring peptides is often referred to as peptidomics. Direct analysis of peptides produced by processing or degradation of proteins might be useful for example for detecting and identifying pathogenic and/or biomarker peptides in body fluids like blood. In this paper, we introduce one of the standard protocols for comprehensive analysis of serum-derived peptides, which consists of methods for purification of serum peptides, detection of peptides, pattern recognition and clustering (bioinformatics), and identification of peptide sequences. Peptide identification should be followed by the investigation of their pathogenic roles using for example synthetic peptides and the establishment of their usefulness as bioclinical markers. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_2 Host–pathogen interactions are complex competitions during which both the host and the pathogen adapt rapidly to each other in order for one or the other to survive. Salmonella enterica serovar Typhimurium is a pathogen with a broad host range that causes a typhoid fever-like disease in mice and severe food poisoning in humans. The murine typhoid fever is a systemic infection in which S. typhimurium evades part of the immune system by replicating inside macrophages and other cells. The transition from a foodborne contaminant to an intracellular pathogen must occur rapidly in multiple, ordered steps in order for S. typhimurium to thrive within its host environment. Using S. typhimurium isolated from rich culture conditions and from conditions that mimic the hostile intracellular environment of the host cell, a native low molecular weight protein fraction, or peptidome, was enriched from cell lysates by precipitation of intact proteins with organic solvents. The enriched peptidome was analyzed by both LC–MS/MS and LC–MS-based methods, although several other methods are possible. Pre-fractionation of peptides allowed identification of small proteins and protein degradation products that would normally be overlooked. Comparison of peptides present in lysates prepared from Salmonella grown under different conditions provided a unique insight into cellular degradation processes as well as identification of novel peptides encoded in the genome but not annotated. The overall approach is detailed here as applied to Salmonella and is adaptable to a broad range of biological systems. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_3 The transparent soil nematode Caenorhabditis elegans can be considered an important model organism due to its ease of cultivation, suitability for high-throughput genetic screens, and extremely well-defined anatomy. C. elegans contains exactly 959 cells that are ordered in defined differentiated tissues. Although C. elegans only possesses 302 neurons, a large number of similarities among the neuropeptidergic signaling pathways can be observed with other metazoans. Neuropeptides are important messenger molecules that regulate a wide variety of physiological processes. These peptidergic signaling molecules can therefore be considered important drug targets or biomarkers. Neuropeptide signaling is in the nanomolar range, and biochemical elucidation of individual peptide sequences in the past without the genomic information was challenging. Since the rise of many genome-sequencing projects and the significant boost of mass spectrometry instrumentation, many hyphenated techniques can be used to explore the “peptidome” of individual species, organs, or even cell cultures. The peptidomic approach aims to identify endogenously present (neuro)peptides by using liquid chromatography and mass spectrometry in a high-throughput way. Here we outline the basic procedures for the maintenance of C. elegans nematodes and describe in detail the peptide extraction procedures. Two peptidomics strategies (off-line HPLC–MALDI-TOF MS and on-line 2D-nanoLC–Q-TOF MS/MS) and the necessary instrumentation are described. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_4 The central nervous systems of molluscan species contain high levels of structurally diverse peptides that function as neurotransmitters, neuromodulators or neurohormones. Peptide diversity is believed to be a way to increase the information handling capacity of neurons in the context of a brain with low cell numbers and neuronal connectivity. Accordingly, much effort has been made to identify peptides from single neurons and tissues of interest. In the past decade a mass spectrometry-based approach has been applied to detect and characterize peptides from single neurons, nerves and tissues of the molluscan brain. Peptides from single neurons are often analysed directly by mass spectrometry without prior sample preparation. Single neurons from the molluscan brain may be identified based on their position, cell morphology and colour. Neurons that cannot be readily identified can be tagged functionally or chemically. For the analysis of peptides from tissues, special extraction methods in conjunction with peptide separation by liquid chromatography coupled to mass spectrometry have been developed. Tens to hundreds of peptides from the tissue extract can be detected and characterized in a single analysis. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_5 Neuropeptides are important signaling molecules that regulate many essential physiological processes. Microdialysis offers a way to sample neuropeptides in vivo. When combined with liquid chromatography–mass spectrometry detection, many known and unknown neuropeptides can be identified from a live organism. This chapter describes sample preparation techniques and general strategies for the mass spectral analysis of neuropeptides collected via microdialysis sampling. Methods for the in vitro microdialysis of a neuropeptide standard as well as the in vivo microdialysis sampling of neuropeptides from a live crab are described. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_6 Spider venom contains a complex mixture of components with a large range of molecular masses (0.1–60 kDa) exhibiting a diverse array of actions. Most of these components are proteinaceous molecules – biologically active proteins and peptides. Proteomics profiling of spider venoms (the components with MW >10 kDa) could be achieved through conventional 2-DE-based proteomics methods combined with MS or MS/MS detection. Peptidomic profiling (of the components with MW below ~10 kDa) is usually achieved through off-line separation by a combination of ion-exchange and reverse-phase chromatography, and it relies more heavily on de novo sequencing by Edman degradation or MS/MS for peptide identification. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_7 Spider venoms represent invaluable sources of biologically active compounds suitable for use in life science research and also having a significant potential for biotechnology and therapeutic applications. The methods reported herewith are based on our long experience of spider venom fractionation and peptides purification. We routinely screen new peptides for antimicrobial and insecticidal activities and our detailed protocols are also reported here. So far these have been tested on species of Central Asian and European spiders from the families Agelenidae, Eresidae, Gnaphosidae, Lycosidae, Miturgidae, Oxyopidae, Philodromidae, Pisauridae, Segestriidae, Theridiidae, Thomisidae, and Zodariidae. The reported protocols should be easily adaptable for use with other arthropod species. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_8 The diversity of insect neuropeptides coupled with the limitations from the small size of the insects themselves combine to make positive identification through peptide sequencing a highly challenging task. The advent of the “soft-ionisation” techniques of MALDI-TOF and electrospray (ESI)-Q-TOF mass spectrometry, coupled with the additional information from insect genome projects have revolutionised the characterisation of insect neuropeptides, such that sequences can now be obtained from just a few cells, where before thousands of insects had to be laboriously dissected, extracted and purified. Some of the procedures that are now used to identify these peptides are described here. Once the neuropeptides have been identified, it then becomes possible to use this knowledge to define physiological functionality. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_9 Direct MALDI-TOF mass spectrometric peptide profiling is increasingly used to analyze the peptide complement in the nervous system of a variety of invertebrate animals, from leech to Aplysia and many arthropod species, especially insects and crustaceans. Proper sample preparation is often the most crucial step to obtain the necessary data. Here, we describe protocols for the use of MALDI-TOF mass spectrometry to directly analyze the peptidome of neuroendocrine tissues of insects, particularly Drosophila melanogaster, by MALDI-TOF MS. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_21 Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarian Hydra is one of the most basal metazoans and is a key model system for studying the peptides involved in these processes. We developed a novel peptidomic approach to the isolation and identification of functional signalling peptides from Hydra (the Hydra peptide project). First, peptides extracted from the tissue of Hydra magnipapillata are purified to homogeneity using high-performance liquid chromatography (HPLC). The isolated peptides are then tested for their ability to alter gene expression in Hydra using differential display-PCR (DD-PCR). If gene expression is altered, the peptide is considered as a putative signalling peptide and is subjected to amino acid sequencing. Following the sequencing, synthetic peptides are produced and compared to their native counterparts by HPLC and/or mass spectrometry (MS). The synthetic peptides, which are available in larger quantities than their native analogues, are then tested in a variety of biological assays in Hydra to determine their functions. Here we present our strategies and a systematic approach to the identification and characterization of novel signalling peptides in Hydra. We also describe our high-throughput reverse-phase nano-flow liquid chromatography matrix-assisted laser desorption ionization time-of-flight mass spectrometry (LC-MALDI-TOF-MS/MS) approach, which was proved to be a powerful tool in the discovery of novel signalling peptides. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_22 The tachykinins represent the largest known peptide family and are responsible for a range of pleiotropic functions in both vertebrates and invertebrates. Recent research has shown a diversity of mechanisms such as mRNA splicing, precursor processing and post-translation modification that can lead to a complex and continually expanding repertoire of tachykinin peptides. The peptidomic analysis of the tachykinins has been hindered by the lack of specific methodologies to capture, purify and characterise each tachykinin. This chapter summarises some of the methods that have been developed in order to further purify and characterise individual groups of tachykinin peptides from the peptidome. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_23 Affinity peptidomics relies on the successfully proven approach used widely in mass-spectrometry-based protein analysis, where protein samples are proteolytically digested prior to the analysis. Unlike traditional proteomic analyses, affinity peptidomics employs affinity detection instead of, or in addition to, the mass-spectrometry detection. Affinity peptidomics, therefore, bridges the gap between protein microarrays and mass spectrometry and can be used for the detection, identification and quantification of endogenous or proteolytic peptides on microarrays and by MALDI-MS. Phage display technology is a widely applicable generic molecular display method suitable for studying protein–protein or protein–peptide interactions and the development of recombinant affinity reagents. Phage display complements the affinity peptidomics approach when the latter is used, e.g. to characterise a repertoire of antigenic determinants of polyclonal, monoclonal antibodies or other recombinantly obtained affinity reagents or in studying protein–protein interactions. 3D materials such as membrane-based porous substrates and acrylamide hydrogels provide convenient alternatives and are superior to many 2D surfaces in maintaining protein conformation and minimising non-specific interactions. Hydrogels have been found to be advantageous in performing antibody affinity assays and peptide-binding assays. Here we report a range of peptide selection and peptide-binding assays used for the detection, quantification or validation of peptide targets using array-based techniques and fluorescent or MS detection. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_24 Polypeptide and protein arrays enable high-throughput screening capabilities for studying molecular interactions and profiling of biomarkers, and provide a powerful functional screening tool for peptidomics. To overcome the limitations of conventional arraying methods, we have exploited cell-free systems for generating arrays of polypeptides by direct on-chip biosynthesis from DNA templates. Here we describe two protocols: (i) Protein In Situ Array (PISA), which allows the generation of polypeptide arrays in a single reaction by spotting cell-free lysate together with PCR DNA on a glass surface pre-coated with a capturing reagent, and (ii) DNA Array to Protein Array (DAPA), which is capable of producing multiple copies of a polypeptide array from a single DNA array template. The main advantage of these methods is in using an in vitro coupled transcription and translation system which circumvents the need to synthesise and purify individual polypeptides. Our methods allow making polypeptide arrays using amplified linear DNA fragments. 2010-01-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-535-4_25 With the entire genome sequence of several animals now available, it is becoming possible to identify in silico all putative peptides and their precursors in an organism. In this chapter we describe a searching algorithm that can be used to scan the genome for predicted proteins with the structural hallmarks of (neuro)peptide precursors. We also describe how to use search strings such as the presence of a glycine residue as a putative amidation site, dibasic cleavage sites, the presence of a signal peptide, and specific peptide motifs to improve a standard BLAST search and make it suitable for searching (neuro)peptides in EST data. We briefly explain how bioinformatic tools and in silico predicted peptide precursor sequences can aid experimental peptide identification with mass spectrometry. 2010-01-08T05:00:00Z