bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sample Preparation for MALDI Mass Analysis of Peptides and Proteins
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L. 
Published: June 2004

AbstractSample preparation may be the most crucial step in mass spectrometric analysis of peptides and proteins by MALDI. The free dipolar ionic peptides or proteins must be incorporated into a crystalline solid solution that is comprised of excess solvent (the matrix), which modulates desorption/ionization by preferentially absorbing the laser energy at the irradiation wavelength and laser pulse intensity being employed. Usually the cocrystalline sample and matrix are prepared by mixing solutions of each component and permitting the mixture to crystallize through evaporation of the solvents. Two methods employing this approach are presented in this unit:
the dried drop method and the rapid evaporation method.
Searching Sequence Databases Over the Internet: Protein Identification Using MS‐Fit
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L. 
Published: June 2004

AbstractPeptide fingerprint mass mapping is widely used for initial identification of proteins separated by gel electrophoresis. This method involves the enzymatic in‐gel digestion of proteins to generate peptides, followed by mass measurement of the cleaved peptides. The experimental mass values are then compared with theoretical values from protein databases, calculated using the cleavage specificity of the enzyme. This unit describes the use of the search program MS‐Fit to identify the closest match or matches.
Enzymatic Approaches for Obtaining Amino Acid Sequence: On‐Target Ladder Sequencing
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L. 
Published: June 2004

AbstractPeptide sequencing by mass spectrometry (MS) is usually based on detecting mass differences associated with various amino acids in the polymer chain. Post‐source decay (PSD) and MS/MS spectra may yield internal peptide sequences. However, determination of the order of the first two, and sometimes the last few, amino acids in the peptide is often problematic without additional experiments. Several enzymatic approaches have proven useful for identifying the N‐ and C‐terminal residues. They involve the use of carboxypeptidases and aminopeptidases to produce peptide ladders for rapid analysis by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). This unit describes the ladder sequence method to generate amino acid sequence information from low‐ to subpicomole quantities of peptides. It can be performed directly on the sample stage, thus minimizing potential sample losses to vials and through sample transfer.
Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using a Nanospray Interface
Authors: Lee Terry D. , Moore Roger E. , Young Mary K. 
Published: June 2004

AbstractElectrospray (ES) is a concentration‐dependent phenomenon, and operation at high flow rates results in a corresponding high rate of sample consumption. Sensitivity increases are most readily achieved by decreasing the flow rate of sample solution into the electrospray source. In recent years, a number of methodologies have been developed to achieve ES analyses at sub‐ml/min flow rates. There are a large number of microscale ES devices currently in use, but this unit describes in detail a micro‐scale interface for direct liquid introduction without separation.
Protein Identification Using a Quadrupole Ion Trap Mass Spectrometer and SEQUEST Database Matching
Authors: Moore Roger E. , Young Mary K. , Lee Terry D. 
Published: June 2004

AbstractA procedure is described for rapid, sensitive protein identification utilizing liquid chromatography‐‐ tandem mass spectrometry. The analysis is performed on mixtures of peptides obtained by enzyme digestion. The SEQUEST computer program is used to match the sequence information in the spectra to a database of known protein sequences.
Introduction to Peptide Synthesis
Authors: Fields Gregg B. 
Published: June 2004

AbstractA number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar‐substitute aspartame to clinically used hormones, such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins, including their purification. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.
Native Chemical Ligation of Polypeptides
Authors: Camarero Julio A. , Muir Tom W. 
Published: June 2004

AbstractThe total synthesis and semisynthesis of proteins allows the site‐specific incorporation of unnatural amino acids, post‐translational modifications, and biophysical/biochemical probes into the target molecule. Among the various chemical and enzymatic approaches available for the synthesis/semisynthesis of proteins, the native chemical ligation technique has proven especially useful and is the exclusive focus of this unit. This unit first discusses how to choose the ligation site(s) in the target protein and then outlines how to obtain the necessary polypeptide building blocks using either chemical synthesis or recombinant DNA expression. Next, the synthesis of a protein by native chemical ligation of two polypeptide fragments is described. The synthesis of a protein from three polypeptide fragments using a sequential native chemical ligation strategy is also described. Support protocols describe how to obtain the necessary polypeptide fragments using either chemical synthesis or recombinant DNA expression.
Detection of Protein‐Protein Interactions by Coprecipitation
Authors: Elion Elaine A. 
Published: June 2004

AbstractCo‐precipitation of proteins from whole cell extracts is a valuable approach to test for physical interactions between proteins of interest. It may be the method of choice, or may be used in conjunction with other procedures to detect protein‐protein interactions, such as two‐hybrid analyis and co‐purification schemes, and tests of physical associations using purified proteins.
Imaging Protein‐Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
Authors: Wouters Fred S. , Bastiaens Philippe I.H. 
Published: June 2004

AbstractDetection of specific protein‐protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein‐protein interactions from the results of these tests. Fluoresence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.
Identification of Protein Interactions by Far Western Analysis
Authors: Edmondson Diane G. , Dent Sharon Y. R. 
Published: June 2004

AbstractThis unit describes far western blotting, a method of identifying protein‐protein interactions. In a far western blot, one protein of interest is immobilized on a solid support membrane, then probed with a non‐antibody protein. As described, far western blots can be used to identify specific interacting proteins in a complex mixture of proteins. They are particularly useful for examining interactions between proteins that are difficult to analyze by other methods due to solubility problems or because they are difficult to express in cells. This method is performed totally in vitro, and the proteins of interest can be prepared in a variety of ways. A protocol is also provided for determining the effects of specific peptide residues or post‐translational modifications on protein‐protein interactions. Many different detection techniques, either radioactive or nonradioactive, can be used. For example, the protein probe may be detected indirectly with an antibody, rather than being labeled radioactively.
Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions
Authors: Cook Neil , Harris Alison , Hopkins Alison , Hughes Kelvin 
Published: June 2004

AbstractScintillation proximity assay (SPA) is a versatile homogeneous technique for radioactive assays which eliminates the need for separation steps. In SPA, scintillant is incorporated into small fluomicrospheres. These microspheres or “beads” are constructed in such a way as to bind specific molecules. If a radioactive molecule is bound to the bead, it is brought into close enough proximity that it can stimulate the scintillant contained within to emit light. Otherwise, the unbound radioactivity is too distant, the energy released is dissipated before reaching the bead, and these disintegrations are not detected. In this unit, the application of SPA technology to measuring protein‐protein interactions, Src Homology 2 (SH2) and 3 (SH3) domain binding to specific peptide sequences, and receptor‐ligand interactions are described. Three other protocols discuss the application of SPA technology to cell‐adhesion‐molecule interactions, protein‐DNA interactions, and radioimmunoassays. In addition, protocols are given for preparation of SK‐N‐MC cells and cell membranes.
Analytical Centrifugation: Equilibrium Approach
Authors: Laue Tom 
Published: June 2004

AbstractThe specific interaction of biological molecules with one another is fundamental to the biochemistry of all living things. Equilibrium sedimentation is a classic method of biochemistry that provides first‐principle thermodynamic information about the molar mass, association energy, association stoichiometry, and thermodynamic nonideality of molecules in solution. It is one of the most rigorous, powerful, and readily adapted methods for characterizing solution interactions.
Analytical Ultracentrifugation: Sedimentation Velocity Analysis
Authors: Stafford Walter 
Published: June 2004

AbstractThe analytical ultracentrifuge is a high speed centrifuge with an optical system allowing observation of the concentration of macromolecules as a function of radius and time. In sedimentation velocity experiments,relatively high speeds are used so that a boundary is formed between the solution of sedimenting macromolecule and the buffer in which it is dissolved. Analysis of the rate boundary movement and evolution of its shape can yield information about the molar masses of species present as well as stoichiometries and equilibrium constants for their interactions. This overview discusses crucial issues pertaining to sedimentation velocity experiments, including non‐interacting and interacting systems, ideality and non‐ideality, and reversible versus kinetically limited equilibrium scenarios.
Overview of Pepsin‐like Aspartic Peptidases
Authors: Dunn Ben M. 
Published: June 2004

AbstractThe aspartic peptidase family of enzymes has been implicated in a variety of disease states, from stomach ulcers, to breast cancer, and even Alzheimer's Disease. This unit describes the major characteristics of the aspartic peptidases, including mechanism of action, subcellular and tissue localization, and biological substrate specificity.
Caspases
Authors: Denault Jean‐Bernard , Salvesen Guy S. 
Published: June 2004

AbstractCaspases are a family of cysteine proteases with a strict specificity for aspartate residues involved in inflammatory process and programmed cell death. This overview unit provides basic information on their structure, enzymatic activity, substrate specificity, activation,inhibition and their implication in pathologies. It is intended to be an overview for investigators that are unfamiliar with this family of enzymes but it is also applicable to scientists pursuing research in this field.
Over‐Expression and Purification of Active Serine Proteases and Their Variants from Escherichia coli Inclusion Bodies
Authors: Parry Marina A. A. 
Published: June 2004

AbstractThis unit describes the over‐expression and purification of active serine proteases and their variants from E. coli inclusion bodies. The strategy includes the folding and purification of a stable zymogen precursor protein, and its later activation with the appropriate convertase to the less stable but active protease. A test to follow the presence of activity in the samples, together with an active‐site titration protocol to determine the number of active sites per mole of total protein are provided. It should be emphasized that although most of the protocols described are applied to a specific example, they are fairly representative of the methods and approaches generally used for laboratory‐scale preparation of other recombinant serine proteases. The critical steps and how this template protocol can be adapted for the purification of other serine proteases are described.
Expression, Purification, and Characterization of Caspases
Authors: Denault Jean‐Bernard , Salvesen Guy S. 
Published: June 2004

AbstractThis unit describes a protocol to obtain milligram amounts of enzymatically active pure recombinant caspases. Specific details for the expression, purification of caspase‐3, ‐6, ‐7, ‐8, ‐9 and ‐10 are discussed along with strategies to obtain particular forms (e.g., the zymogen) of some of them.
Zymography of Metalloproteinases
Authors: Troeberg Linda , Nagase Hideaki 
Published: June 2004

AbstractZymography is an electrophoretic technique enabling visualization of the number and approximate size of peptidases in a sample on the basis of their hydrolysis of a protein substrate within the gel. The technique is particularly useful for analyzing the peptidase composition of complex biological samples because visualization depends directly on proteolytic activity. This unit presents a representative zymography protocol for the study of matrix metallopeptidases (MMPs).
Protein Profiling Using Two‐Dimensional Difference Gel Electrophoresis (2‐D DIGE)
Authors: Lilley Kathryn S. 
Published: June 2004

Abstract2D‐DIGE relies on pre‐electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one gel. The dye‐labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with spot matching between gels. Image analysis programs can then be used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable expression differences to be identified and quantified. This unit describes the DIGE procedure in terms of sample preparation from various types of cells, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples.
Laser Capture Microdissection for Proteome Analysis
Authors: Craven Rachel A. , Banks Rosamonde E. 
Published: June 2004

AbstractLaser capture microdissection is being employed increasingly to isolate specific cell types from tissues, thus overcoming problems of experimental interpretation due to tissue heterogeneity of samples. This unit describes protocols which have been optimized to allow laser capture microdissection of tissues with minimal effect on protein integrity, and their subsequent analysis by techniques including 2D‐PAGE, immunoblotting and SELDI, and discusses the relative merits of this approach.
Preparing Protein Extracts for Quantitative Two‐Dimensional Gel Comparison
Authors: Chevallet Mireille , Tastet Christophe , Luche Sylvie , Rabilloud Thierry 
Published: June 2004

AbstractThis unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample‐dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for sample‐preparation optimization. These solubilization procedures take into account the constraints brought by two‐dimensional electrophoresis and are thus well suited for proteomic approaches.
Analysis of Protein Composition Using Multidimensional Chromatography and Mass Spectrometry
Authors: Link Andrew J. , Jennings Jennifer L. , Washburn Michael P. 
Published: June 2004

AbstractMultidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in‐gel digestions by directly identifying protein mixtures in solution.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C. 
Published: June 2004

AbstractThe first section of this appendix unit discusses basic principles of sterile technique, while the second section discusses medium preparation. A protocol is then provided for subculturing of a monolayer culture grown in petri dishes or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An alternate protocol describes freezing suspension cells.
Quantitation of Nucleic Acids with Absorption Spectroscopy
Authors: Gallagher Sean 
Published: June 2004

AbstractThis appendix describes a protocol for measuring absorption of nucleic acid samples at several different wavelengths to assess purity and concentration. A260 measurements are quantitative for relatively pure nucleic acid preparations in microgram quantities. Absorbance readings cannot discriminate between DNA and RNA; however, the ratio of
A at 260 and 280 nm can be used as an indicator of nucleic acid purity.
Theoretical Aspects of the Quantitative Characterization of Ligand Binding
Authors: Sawyer William H. , Winzor Donald J. 
Published: June 2004

AbstractLiving organisms grow, differentiate, reproduce, and respond to their environment via specific and integrated interactions between biomolecules. The investigation of molecular interactions therefore constitutes a major area of biochemical study, occupying a ubiquitous and central position between molecular physiology on the one hand and structural chemistry on the other. While specificity resides in the details of structural recognition, the dynamic interplay between biomolecules is orchestrated precisely by the thermodynamics of the biomolecular equilibria involved. A common set of physicochemical principles applies to all such phenomena, irrespective of whether the interaction of interest involves an enzyme and its substrate or inhibitor, a hormone or growth factor and its receptor, an antibody and its antigen, or, indeed, the binding of effector molecules that modulate these interactions. The binding affinity, binding specificity, number of binding sites per molecule, as well as the enthalpic and entropic contributions to the binding energy are common parameters that assist an understanding of the biochemical outcome. This unit aims to provide an overview of the design and interpretation of binding experiments.
Practical Aspects of Radioligand Binding
Authors: McKinney Michael 
Published: June 2004

AbstractRadioligand binding assays can be used to characterize receptors in their natural environment (wild type) as well as those transfected into cell lines. They can be used to study receptor dynamics and localization, to identify novel chemical structures that interact with receptors, and to define ligand activity and selectivity. The technique thus serves as a valuable adjunct to other pharmacological and molecular biological approaches. This unit describes the major issues involved in developing a binding assay, such as selecting a radioligand, tissue preparation, optimization of assay parameters, and separation of bound from free ligand. Methods for analysis of the binding data are carefully considered, and tips for troubleshooting a binding assay are also provided.
Characterization of Adrenoceptors
Authors: Bylund David B. 
Published: June 2004

AbstractNorepinephrine and epinephrine are important neurotransmitters in both the peripheral and the central nervous systems. The actions of these neurotransmitters are mediated by adrenoceptors. Three major types of adrenoceptors have been identified on the basis of pharmacological data: alpha1, alpha2, and beta. Within each major type, at least three receptor subtypes have been identified. Three basic types of radioligand binding experiments are provided in this unit: (1) saturation binding from which the affinity (Kd) of the radioligand for the adrenoceptor and the binding site density (Bmax) can be determined; (2) inhibition experiments from which the affinity (Ki) of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic assays from which the forward and reverse rate constants of the binding process can be determined. Three support protocols are provided covering the preparation of membranes containing the receptor from tissue or cells in culture, calculation of Kd and Bmax from saturation experiments, and calculation of Ki from inhibition experiments.
Characterization of Dopamine Receptors
Authors: Levant Beth 
Published: June 2004

AbstractDopamine receptors are the principal targets of drugs used for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson's disease. This unit details protocols for radioligand binding assays for various subtypes of dopamine receptor. The scope is limited primarily to the most widely used, well‐established protocols for binding assays, which employ commercially available tritiated ligands. Protocols are given for radioligand binding assays for D1‐like and D2‐like receptors. A protocol for the putatively selective labeling of D3 sites is also presented. Although the basic protocols are used to assay dopamine receptors in membrane homogenates, support protocols describe the preparation of these homogenates from organs and from cultured cells. The calculation of the radioligand's molar concentration is also included.
Characterization of Neuronal Nicotinic Acetylcholine Receptors
Authors: Sullivan James P. , Anderson David J. 
Published: June 2004

AbstractNeuronal nicotinic acetylcholine receptors are members of the ligand‐gated ion channel superfamily of receptors. The potential of nAChRs as targets of therapeutic intervention has been revealed, in part, by recent studies showing that a diversity of nAChR subunits exist in brain and other tissues, and that discrete subunit combinations may be involved in mediating specific neurophysiological functions and behaviors. To date, eleven gene products representing nAChRs have been identified. This unit examines the binding properties of the a4 b2 receptor in rat brain or in transformed cell lines expressing the recombinant receptor.