bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gel Electrophoresis in Protein and Peptide Analysis
Authors: Kaplan Batia 
Published: September 2006

AbstractGel electrophoresis is a widely used method in biochemical research; a number of different forms of gel electrophoresis have been developed and applied to the analysis of proteins and peptides. The most popular form is polyacrylamide gel electrophoresis (PAGE), in which proteins are separated within a gel matrix on the basis of differences in their charge density and/or relative molecular mass (Mr). Polyacrylamide gel is also widely used as a media for the generation of pH gradients in an isoelectrofocusing (IEF) technique, where separation of proteins is due to their differences in isoelectric point (pI). Two‐dimensional (2D) electrophoresis is an extremely powerful separation technique, allowing differentiation of proteins on the basis of their pI in IEF, and their Mrin sodium dodecyl sulfate (SDS)/PAGE. Agarose gels are rarely used today in the electrophoretic analysis of proteins; their utility is mainly restricted to the immunoelectrophoretic techniques, which allow the characterization of proteins by their migration in gel and immunological properties.This article provides a brief history of gel electrophoresis and describes the principles of major electrophoretic techniques. The utility of gel electrophoresis in different fields of biochemical research is also demonstrated. Topics included are analytical and small‐scale preparative separations of complex proteins samples; determination of molecular mass of the electrophoretically separated proteins and polypeptides; electrophoretic micropreparation of a protein sample for its further immunochemical, enzymatic, and chemical analysis; study of protein interactions with different ligands; examination of protein unfolding and its quaternary structure. The advantages and limitations of the described techniques are discussed.
High‐Performance Liquid Chromatographic Separations and Equipment in Peptide and Protein Analysis, Miniaturization of
Authors: Nice E.C. , Catimel B. 
Published: September 2006

Abstract
The use of short narrow‐bore (2.1‐mm i.d.), microbore (1‐mm i.d.) or capillary (<1‐mm i.d.) columns allows the recovery of purified proteins and peptides in reduced volume at concomitantly increased concentration compared with larger bore columns. Additionally, sample detection is correspondingly improved for concentration‐dependent detectors. Sample volumes are not limiting: large sample volumes can be loaded on to interactive supports (e.g. reversed‐phase (RP), ion‐exchange (IEX), hydrophobic interaction (HI), affinity) under appropriate mobile phase conditions (trace enrichment). Suitable instrumentation requires careful consideration with regard to minimized pre‐ and postcolumn dead volumes, accurate gradient formation at low flow rates, detector flow cell geometry and sample recovery. These techniques are ideally suited for the purification of trace protein or peptide components in bulk biological samples prior to application to highly specific and sensitive downstream analytical techniques (e.g. microsequence analysis, mass spectrometry (MS), biosensor analysis, biological assay). They also facilitate the recovery of low levels of material following chemical or enzymatic modification (e.g. radioiodination, enzymatic semi‐synthesis, chemical derivatization). The techniques can also be applied to the development of large‐scale processes, whereby multidimensional purification protocols can be rapidly developed using minimum amounts of sample, solvent and column packing materials and then scaled up appropriately.
High‐Performance Liquid Chromatography/Mass Spectrometry in Peptide and Protein Analysis
Authors: Shen Tun‐Li 
Published: September 2006

Abstract
Since the 1970s, high‐performance liquid chromatography/mass spectrometry (HPLC/MS) has gone through many stages of development. The main technical challenge has been the coupling of liquid chromatography (LC) systems with mass spectrometers. Soft ionization techniques that have contributed to HPLC/MS for peptide and protein study include fast atom bombardment (FAB), matrix‐assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI). Among these techniques, FAB was developed first and used extensively in the 1980s; it lacked the sensitivity of the other two more recent techniques. MALDI and ESI are comparable in terms of sensitivity and the types of biomolecules that can be analyzed. However, MALDI has the slight advantage of being more tolerant to salts and able to analyze complex mixtures. On the other hand, the ability of ESI to interface mass spectrometry (MS) directly with LC makes it extremely useful for protein and peptide analysis. Another advantage that ESI offers is its ability to produce multiply charged protein ions, which allows the observation of large proteins with mass analyzers of limited mass range. In this article we discuss the experimental parameters that are important in HPLC/MS and the considerable progress made in ESI source design and capillary HPLC/MS.

In the study of natural and synthetic peptides, HPLC/MS has been used to separate, characterize, and sequence the peptides being investigated. In protein analysis, HPLC/MS is most useful in characterizing protein post‐translation modification such as glycosylation and phosphorylation. We discuss specific HPLC/MS methods for detecting glycopeptides and phosphopeptides during chromatographic separation by monitoring marker ions: Hex
+
(m/z 163), HexNAc
+
(m/z 204), NeuAc
+
(m/z 292), HexHexNAc
+
(m/z 366) for glycopeptides, and PO
2
−
(m/z 63), PO
3
−
(m/z 79) for phosphopeptides. Another important application of HPLC/MS is in proteomic analysis. Here, the most significant development in the last decade has been the implementation of MS as the primary analytical technique to identify proteins separated by two‐dimensional polyacrylamide gel electrophoresis (2D/PAGE). Additionally, the need to identify gel‐separated proteins has promoted various MS‐based protein database search strategies. These include peptide mass mapping using proteolytic peptide masses for database search, and using peptide fragmentation data obtained from tandem mass spectrometry (MS/MS) to search protein sequence databases. Finally, we examine the recent advance in integrating HPLC/MS and database search strategy in proteomic analysis.
Hydrophilic‐Interaction Chromatography in Peptide and Protein Analysis
Authors: Jenö Paul 
Published: September 2006

AbstractHydrophilic‐interaction liquid chromatography (HILIC) has been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. A hydrophilic stationary phase is eluted with a hydrophobic mobile phase whereby retention of solutes increases with the hydrophilicity of solutes. Bound solutes are eluted by decreasing the hydrophobicity of the mobile phase. Mixed mode effects based on hydrophilic and ionic interactions can also be exploited by proper selection of the stationary and the mobile phase. Typically, the order of elution is the opposite of that obtained by reversed‐phase chromatography (RPC). HILIC is particularly interesting for solutes which are difficult to chromatograph on conventional reversed‐phase materials. This review focuses on the application of HILIC to the separation of amphipathic α‐helical peptides, the analysis of protein glycosylation, phosphorylation, and acetylation, and to the removal of detergents from membrane proteins.
Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry in Peptide and Protein Analysis
Authors: Lewis J. Kathleen , Wei Jing , Siuzdak Gary 
Published: September 2006

Abstract
Matrix‐assisted laser desorption/ionization mass spectrometry (MALDIMS), first introduced in 1988 by Hillenkamp and Karas, has become a widespread analytical tool for peptides, proteins and most other biomolecules. In MALDI (matrix‐assisted laser desorption/ionization) analysis, the analyte is first co‐crystallized with a large molar excess of a matrix compound, usually an ultraviolet (UV)‐absorbing weak organic acid, after which laser radiation of this analyte–matrix mixture results in the vaporization of the matrix which carries the analyte with it. The matrix therefore plays a key role by strongly absorbing the laser light energy and causing, indirectly, the analyte to vaporize. The matrix also serves as a proton donor and receptor, acting to ionize the analyte in both positive and negative ionization modes, respectively. The efficient and directed energy transfer during a matrix‐assisted laser‐induced desorption event provides high ion yields of the intact analyte and allows for the measurement of compounds with high accuracy and subpicomole sensitivity. The ability to generate such accurate information can be extremely useful for protein identification and characterization. For example, a protein can often be unambiguously identified by the accurate mass analysis of its constituent peptides (produced by either chemical or enzymatic treatment of the sample). This article discusses basic MALDI concepts and instrumentation and focuses on applications in the field of peptides and proteins, specifically on the utility of MALDI in protein identification, protein structural studies, and as a clinical assay.
Molecular Modeling in Peptide and Protein Analysis
Authors: Kuczera Krzysztof 
Published: September 2006

AbstractThis article describes the theoretical background and practical applications of classical molecular mechanics and molecular dynamics (MD). Following a brief historical outline of molecular modeling, the concept of the molecular potential energy function is introduced and the basic algorithms of molecular mechanics: energy minimization, normal mode analysis, MD and Monte Carlo (MC) simulations are defined. The advantages of molecular modeling methods – speed of calculations and detailed microscopic insight – are described. The shortcomings – approximate nature and difficulties with conformational sampling – are also analyzed. A number of modern approaches aimed at improving the reliability of computer simulations are briefly presented, including advanced energy optimization methods, treatment of long‐range forces and accelerated conformational exploration. Examples of applications of molecular modeling methods to study of various aspects of structure, dynamics and function of peptides and proteins are presented. A brief exposition of the relationship between molecular modeling and some fundamental experimental techniques, such as X‐ray crystallography, neutron scattering, nuclear magnetic resonance (NMR), fluorescence and vibrational spectroscopies is provided. Finally, an outline of future directions of modeling studies is suggested.
Peptide Diastereomers, Separation of
Authors: Scriba Gerhard K.E. 
Published: September 2006

Abstract
Diastereomeric peptides, isomers in which one or more of the chiral centers have been converted to the opposite configuration (R or S), often possess different biological activities and/or conformational properties. Therefore, the separation and determination of diastereomeric peptides is particularly significant to the pharmaceutical industry for quality control of peptide synthesis and stability as well as for regulatory requirements. Diastereomers differ in their physicochemical properties which can be utilized for their determination by numerous techniques. In contrast to many other methods such as optical rotation, circular dichroism, differential scanning calorimetry, infrared (IR) spectroscopy, or nuclear magnetic resonance (NMR) spectroscopy only chromatographic techniques and capillary electrophoresis (CE) allow the sensitive and simultaneous identification and quantification of peptide diastereomers.
The current chapter will cover the analytical scale separation of peptide diastereomers by chromatographic methods, including paper chromatography (PC), thin‐layer chromatography (TLC), gas chromatography (GC), and high‐performance liquid chromatography (HPLC) as well as CE. HPLC is currently the predominant method for peptide diastereomer separations. However, due to the high resolving power particularly for polar compounds CE has been applied to the separation of diastereomeric peptides in recent years. This article will cover methods employing achiral stationary phases or buffers without chiral additives for the separations. Moreover, only examples of diastereomeric peptides containing natural amino acids will be discussed.
Surface Plasmon Resonance Spectroscopy in Peptide and Protein Analysis
Authors: Salamon Zdzislaw , Tollin Gordon 
Published: September 2006

Abstract
This chapter presents a detailed overview of the theoretical basis of surface plasmon resonance (SPR) spectroscopy, emphasizing both principles and practical considerations such as thin‐film designs for SPR devices which can be employed in characterization of biological materials, including peptides and proteins. It is demonstrated that the analysis of the resonance spectrum yields structural parameters of anisotropic films of interacting biomolecules, including distribution of mass, long‐range molecular order, and molecular conformational changes. Binding parameters, including binding affinities and rate constants, can also be determined. A summary is presented of different techniques for biomolecule immobilization on SPR‐generating surfaces applicable in peptide and protein analysis.
Chiral Pesticides and Polychlorinated Biphenyl Congeners in Environmental Samples, Analysis of
Authors: Garrison Arthur W. 
Published: September 2006

AbstractOver 25% of pesticides and other toxic organic pollutants are chiral, as are 19 of the 209 polychlorinated biphenyl (PCB) congeners; that is, they exist as two mirror image species called enantiomers (PCB enantiomers are called atropisomers). The enantiomers of a chiral compound have identical physical and abiotic chemical properties, but usually differ in biological properties such as microbial degradation, uptake and transport across membranes, metabolism rate, and toxicity. Chirality in the environment has become recognized as an important phenomenon since the mid‐1980s, and related research has centered on the need to understand the environmental fate and effects of pesticides and PCBs. This article provides a brief background on chiral chemistry in general and the importance of chirality in the drug industry, followed by a summary of examples of the enantioselectivity of pesticide and PCB occurrences and degradation in the environment as gleaned from the scientific literature. The main emphasis is on the analysis of these compounds, with the primary focus on chiral separations. Chiral separations are generally performed by gas chromatography (GC) or high‐performance liquid chromatography (HPLC) using chiral columns, or, to a lesser extent, by capillary electrophoresis (CE) using cyclodextrin (CD) or other chiral selectors in the run buffer. Summaries of these three separation methods are provided, with figures giving examples of pesticide and PCB enantiomeric separations. Finally, to show the usefulness of these separation methods, applications to several environmental chemistry problems involving chiral pesticides and PCBs are described and illustrated.
High‐Performance Liquid Chromatography Methods in Pesticide Residue Analysis
Authors: Hogendoorn Elbert A. 
Published: September 2006

AbstractThe important role of high‐performance liquid chromatography (HPLC) in pesticide residue analysis (PRA) is presented. Based on the experience and view of the author, a number of reliable and robust liquid chromatography (LC) approaches and methods have been selected to highlight the favorable features of this technique in this field of analysis.Reversed‐phase liquid chromatography (RPLC) with ultraviolet (UV) detection mode is most widely adopted, owing to its ability to perform efficient separation of very polar to apolar pesticides and the universal character of the detection mode. Therefore, a large part will deal with reversed‐phase liquid chromatography/ultraviolet (RPLC/UV) applications showing its excellent separation performance and detectability of compounds not directly amenable to gas chromatography (GC).Another important feature of RPLC is the possibility of including sample pretreatment steps such as sample concentration and/or cleanup in the analytical procedure. The very good compatibility with aqueous samples or extracts easily permits RPLC on‐line sample loading (trace enrichment) by means of column switching, allowing fully automated procedures. It will be shown that, depending on whether the first column is a low‐ or a high‐resolution column, two different modes of column switching, solid‐phase extraction (SPE) to LC and LC to LC, are adopted in the determination of pesticides. The type of columns and materials, the advantages and the application range of both column‐switching modes is discussed in detail. Attention will be paid to method development including both the straightforward trial‐and‐error approach and more systematic method development procedures.The more selective/sensitive detection modes, fluorescence detection (FD) and mass spectrometry (MS) as applied in pesticides residue analysis will be discussed with emphasis on the increasing importance of MS for identification and quantification of polar pesticides in various types of samples.Finally, the use of LC methods involving normal‐phase liquid chromatography (NPLC), low‐resolution RPLC and gel permeation chromatography (GPC), will demonstrate the powerful sample pretreatment feature of LC prior to gas chromatographic analysis.
Immunochemical Assays in Pesticide Analysis
Authors: Dankwardt Andrea 
Published: September 2006

AbstractImmunochemical assays (immunoassays, IAs) are biochemical assays which work according to the law of mass action. They are based on the recognition of an antigen (Ag) or a hapten by antibodies (Abs). Abs are serum glycoproteins of the immunoglobulin (Ig) class and are produced by the vertebrate immune system against foreign material of high molecular mass. The result of the binding reaction between the Ab and an analyte is usually made visible by means of enzymatic, chemiluminescent, fluorescent or radioactive markers. According to the label used IAs can be classified into enzyme immunoassays (EIAs), radioimmunoassays (RIAs), fluorescence immunoassays (FIAs) or chemiluminescent immunoassays (CLIAs). The measuring range of most IAs for pesticides is in the parts per trillion to lower parts per billion range. A lot of samples can be analyzed within a short time, while only low sample volumes are necessary. In many cases (water, some liquid food samples) no extraction step and no cleanup are necessary. Not all assays are completely specific to one single compound. Cross‐reactivities of the Abs with haptens similar to the analyte can be observed. In some cases, matrix effects may occur, especially with soil or colored food extracts. Therefore, validation of the assays for the matrix of interest should be carried out. As IAs are usually targeted at a single analyte or a group of analytes, multianalyte approaches using Ab arrays or a combination of immunochemical techniques with liquid chromatography (LC) are pursued.
Multiclass, Multiresidue Analysis of Pesticides, Strategies for
Authors: Lehotay Steven J. 
Published: September 2006

AbstractStrategies for multiclass, multiresidue analysis of pesticides depend on several factors that all derive from three fundamental considerations: (1) the need for the results; (2) the form and composition of the matrix; and (3) the time, resources, and technology available to the analyst. First, the need for the data dictates the quality assurance (QA)/quality control (QC) guidelines and the desired analytical figures of merit, such as limits of detection (LOD) and the accuracy and precision of the results. These needs also contribute to the choice of pesticide analytes, which sets the polarity range for the analysis. No single method can determine all pesticides in diverse matrices, so an overall strategy is often used to compartmentalize a series of approaches in order best to detect the targeted pesticides in an effective and efficient process. Second, the various types of matrices that are commonly analyzed for pesticide residues may affect the sample preparation approach employed in the analytical strategy. Common matrices for pesticide analysis consist of solids, liquids, and gases in applications associated with food, agricultural, environmental, and other sample types. Varying degrees of pesticide–matrix interactions in different materials require more or less stringent extraction procedures to separate the pesticides from the sample. Sample preparation in multiclass, multiresidue strategies frequently utilizes liquid–solid, liquid–liquid and other partitioning processes including those, such as pressurized fluid extraction (PFE), in which pressure and temperature of different fluids are controlled. Extracts often require cleanup to remove interfering co‐extracted matrix components prior to analysis. A variety of cleanup techniques based on a number of partitioning processes, such as liquid chromatography (LC), may be employed in an on‐line or off‐line fashion. Lastly, the sample extract is usually further separated and analyzed using one or more techniques. The majority of pesticides are analyzed by gas chromatography (GC) with mass spectrometry (MS) and/or element‐selective GC detectors. High‐performance liquid chromatography (HPLC) is often used in the analysis of thermally labile pesticides that are not amenable to GC analysis. Other alternatives for analysis of certain pesticide include capillary electrophoresis (CE) and immunoassays. Fundamentally, a high degree of selectivity helps in generating excellent analytical results, but the wide polarity range of analytes in multiclass, multiresidue methods (MRMs) for pesticides necessitates a great deal of effort to attain a high degree of selectivity. Trade‐offs and compromises are omnipresent in multiclass, multiresidue analysis of pesticides, and a host of strategies are available depending on how the analytical chemist prioritizes the analytical and practical considerations.
Organophosphorus Pesticides in Water and Food, Analysis of
Authors: Lacorte Sílvia , Barceló Damià 
Published: September 2006

AbstractThe purpose of this article is to provide an overview of the main characteristics of organophosphorus pesticides (OPPs), such as their physicochemical characteristics, sources, fate and distribution in the environment. It covers regulatory compliance from the Commission of the European Communities (CEC) and the United States National Pesticide Survey (USNPS), regarding the maximum permissible levels of the most toxic and persistent OPPs and their transformation products (TPs) in water. This article also describes the analytical methods currently used to monitor pesticides and their degradation products in water and food matrices. A major part concerns sample preparation techniques and includes traditional liquid–liquid extraction (LLE) and novel techniques based in automated solid‐phase extraction (SPE), either on‐line or off‐line. New sorbent materials, their applications and the parameters involved in the extraction are described. Chromatographic methods used for their determination are discussed in detail. Much emphasis has been given to gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS) as a confirmation technique and mass spectral information on several pesticides has been included. The final section is devoted to method validation, with the aim of providing a basis and a framework of information for quality assurance (QA) and interlaboratory calibration studies.