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Hydrophobic interaction chromatography (HIC) is a column chromatographic separation technique frequently used for the purification of macro-molecules such as proteins and polynucleotides. Purification schemes often are improved by incorporating HIC along with ion exchange, size exclusion, and affinity chromatography HIC has found wide use for the purification of membrane proteins (1), serum proteins (2-4) nuclear proteins, polynucleotides (5), receptors (6,7), cells (8,9) and recombinant proteins (10-12). [ABSTRACT FROM AUTHOR]

A major goal of proteomics is qualitative and quantitative analysis of all the proteins expressed in a cell, tissue, or organism. Changes in protein expression owing to a stimulus or condition are measured in a systematic manner, and are used to elucidate mechanisms of cell function and signaling. A strength of proteomics is that a "shot-gun" approach requiring no prior knowledge of the system is often used and does not assume a model prior to data collection. Therefore, proteomics provides the ability to deal with the complexity of biological systems with minimal experimental bias. The complexity of biological systems arises from the numerous parallel signaling pathways that interact with each other. The ability to monitor many proteins simultaneously yields a global view of protein expression and posttranslation modifications, which is much more informative than monitoring a few proteins. Methods that follow a few proteins and assume a model are more likely to miss interactions and yield biased results. [ABSTRACT FROM AUTHOR]

Combinatorial chemistry (1-7) has evolved from the synthesis of very large mixtures to the preparation of collections of isolated compounds by parallel syntheses. Peptides constitute ideal materials because their preparation is completely automated using solid-phase methodology allowing high throughput syntheses. [ABSTRACT FROM AUTHOR]

High-performance liquid chromatography (HPLC) can be used in enzymatic analysis in order to achieve a good purification of the reaction product. In this chapter, we will see how to use HPLC to measure histamine formation, a process catalyzed by the enzyme histidine decarboxylase (EC 4.1.1.22). Often the tissue under analysis is a poor source of this enzyme, expressed only by a few cell types such as histaminergic neurons, mast cells, and gastrointestinal enterochromaffin-like cells. For this reason, a method of high sensitivity is needed to determine histamine synthesis in tissue slices or histidine decarboxylase activity in low homogenate volumes. However, it may be difficult to eliminate the precursor histidine from histamine purifications because of their similar chemical properties. The method we describe here is based on an HPLC purification of histamine (1) that eliminates histidine much more selectively than previous methods based on cation-exchange gravity columns (2-5). A radiolabeled substrate is used to obtain to the maximum sensitivity and specificity of the assay. [ABSTRACT FROM AUTHOR]

Immunization with a foreign antigen causes B cells of the immune system to produce antibodies of exquisite specificity toward the challenging antigen. This specific reactivity has made antibodies an essential tool for the detection and purification of protein in all fields of biological research. IgG is the most predominant class of serum antibody and is an integral part of many applications within the laboratory. The need to purify monoclonal or polyclonal antibodies is largely determined by the intended application of the antibody. Unpurified antibody is well suited to use in indirect flow cytometry assays, most enzyme-linked immunosorbent assays (ELISAs), for cytotoxicity assays or Western blot analyses. Purified antibody must be used, however, when accurate concentrations are required, chemical modifications such as labeling with fluorescent or radioactive probes are needed, when fragmentation of the antibody is required for binding or crystallization analysis or when antibody is directly coupled to a matrix for immunoaffinity chromatography. [ABSTRACT FROM AUTHOR]

Many cellular processes are ultimately regulated by protein: DNA interactions at the level of transcription. It is increasingly evident that accessing the information stocked in the genetic code is in itself an intrinsic part of the regulation of gene expression. The basal transcription machinery consists of a promoter sequence that is recognized by its cognate RNA polymerase and begins to processively copy the template strand of the DNA into RNA (1,2). Evolution has modeled this process so as to enable modulation at one or more of a number of control points. In most cases, this involves the participation of accessory proteins that bind to specific DNA sequences and affect either the initial recruitment of RNA polymerases or ensuing isomerisations and rearrangements that lead to a functional transcription complex. [ABSTRACT FROM AUTHOR]

Ion-exchange chromatography (IEX) separates biomolecules (proteins, polypeptides, nucleic acids, polynucleotides, charged carbohydrates, and polysaccharides) based on differences in their charge. IEX can be a highly selective chromatographic technique, being able to resolve, for example, proteins which differ by only a single charged group (1). The process relies upon the formation of ionic bonds between the charged groups on biomolecules (typically, −NH3+, =NH2+, ≥NH+, −COO−, PO4−, SO32−), and an ion-exchange gel/support carrying the opposite charge. Non-bound biomolecules (i.e., neutral molecules which do not carry any electrical charge, or molecules carrying the same charge as the ion-exchange support) are removed by washing, and bound biomolecules are recovered by elution with a buffer of either higher ionic strength, or altered pH. The advantages of IEX are 1) high resolving power, 2) separations can be fast, 3) in general, recoveries are high, 4) buffer components are nondenaturing and frequently compatible with further downstream chromatographic separation or assay systems, 5) process can be used as a concentration step, to recover proteins from a dilute solution. The disadvantages of IEX are few, but include 1) the sample must be applied to the IEX support under conditions of low ionic strength and controlled pH, which sometimes requires an extra buffer exchange step to be inserted, 2) chromatographic instrumentation should be resistant to salt-induced corrosion, and 3) postchromatographic concentration of dilute solutions of recovered proteins can result in high salt concentrations (>1 M), unsuitable, for example, in biological assays unless buffer exchange is carried out. [ABSTRACT FROM AUTHOR]

High-performance liquid chromatograpghy (HPLC) separations of membrane proteins can be conveniently divided into two categories. First, there are many methods available for isolation of functional membrane proteins. Typically, the proteins are maintained in configurations as close to their native state as possible through the use of mild detergents that provide solubility without denaturation, allowing convenient ion-exchange or size-exclusion chromatography, for example. Proteins or complexes isolated in this way are subsequently used for functional analysis or crystallization, and so on. These isolation techniques have been well reviewed and readers are referred to literature specific to the protein of interest. The second category of separations are those used to separate membrane proteins from detergents and salts for the purpose of protein chemistry; although tempting to call these methods "denaturing" there is substantial evidence that this is not always the case. The focus of this chapter is to review the latter category of HPLC techniques with specific reference to those methods that provide conditions compatible with mass spectrometric analysis, especially on-line electrospray ionization. [ABSTRACT FROM AUTHOR]

Venoms have attracted significant study in recent years as a reservoir of complex libraries of natural products possessing a wide range of biological activities. Moreover, venoms contain specific and potent molecules that may be utilized in pharmaceutical development and in the production of environmentally friendly insecticides. The compositions of venoms are typically highly complex and contain a variety of molecules including proteins, peptides, and numerous types of small molecules. This complexity requires highly sensitive techniques to allow separation of these components for study. The techniques should also be able to accommodate large variations in sample size to account for the differences in venom available from different creatures (e.g., some snakes can supply up to 500 mg of crude venom from a single milking, whereas some small insects, such as ants, supply submicrogram amounts [1]). These qualities have been found and continue to be advanced in the technique high-performance liquid chromatography (HPLC), in particular reversed-phase HPLC (RP-HPLC). This technique combined with a variety of detection methods can allow the collection of a significant amount of data from very small venom samples. [ABSTRACT FROM AUTHOR]

The human major histocompatibility complex (MHC) is located on the short arm of chromosome 6 and encompasses approx 4 Mb, or 0.1%, of the genome. This region is, by far, the most polymorphic of the human genome. More than 220 genes have been identified in this region and at least 10% of these genes have a direct function in immune responses. The human MHC can be divided into three regions that encode the class I, class II, and class III human leukocyte antigen (HLA) gene products. These HLA molecules demonstrate tremendous polymorphism, which reflects the natural evolution of these genes in response to various microbial pathogens in different ethnic populations. HLA class I molecules are expressed on all nucleated cells and associate with short peptides (8-11 amino acids in length) derived from both self and foreign antigens. These peptide ligands are primarily generated in or transported into the cytoplasm and subsequently translocated into the endoplasmic reticulum (ER) where they assemble with nascent MHC class I molecules. These mature, peptide-loaded, complexes are then transported to the cell surface where they are scrutinized by CD8+ cytotoxic T lymphocytes (CTL). Should the peptide ligand be derived from a pathogen and be recognized as foreign in an immunocompetent host, the cell is killed via the cytotoxic armory of the CTL. The expression of HLA class II molecules is confined to a small subset of highly specialized cells called antigen-presenting cells (APCs). [ABSTRACT FROM AUTHOR]

Bioactive peptides are generally synthesized within large precursor molecules, from which the active moiety must be enzymatically excised by one or more specific peptidases (1). For most peptides, this processing occurs intracellularly, within the secretory pathway, but cosecretory or extracellular processing can also occur. Peptidases also play a critical role in the termination of peptide signals, via cleavage to inactive fragments (2,3). The characterization of the peptidases involved in the generation and metabolism of peptides is thus of critical importance to the understanding of the physiology of specific peptide hormones and neurotransmitters. [ABSTRACT FROM AUTHOR]

A variety of recombinant glycoproteins, including erythropoietin (EPO), and tissue plasminogen activator have been developed as medical agents. The carbohydrate moieties are known to be implicated in the biological activity, metabolic fate, stability, and solubility of these compounds, and it is, therefore, important to analyze the structural features of carbohydrate moieties as well as polypeptide chains in glycoprotein products (1). [ABSTRACT FROM AUTHOR]

In higher organisms, the majority of proteins are posttranslationally modified at some stage, very often resulting in an essential change in the function of the protein. Some modifications change the protein solubility, others are used as molecular switches and thus modify biological activity, whereas others are used to locate proteins to different cell compartments (e.g., ref. 1). Because a given modification results in a change in the molecular mass of the affected amino acid, mass spectrometry (MS) with its unique sensitivity, high mass accuracy, and its ability to deal with complex mixtures, is the method of choice for characterization of posttranslational modifications (2-4). [ABSTRACT FROM AUTHOR]

Analysis of intact proteins is hampered by the fact that even when using high-resolution techniques, like electrospray mass spectrometry, you will only be able to tell whether the protein has the expected mass. If you find a deviation from the expected you will, in most cases, be left wondering where (and perhaps what) the difference is. These observations are compounded when using low-resolution techniques like gel filtration or sodium dodecyl sulfate (SDS) gel electrophoresis. Although 2D gel electrophoresis is able to show a single charge difference, in the absence of additional information you are still left wondering where and what the differences are. [ABSTRACT FROM AUTHOR]

The scale-up of a chromatographic process from the laboratory involving the processing of milliliters of material, to pilot and manufacturing scale involving the processing of liters or hundred liters of material, requires chromatographic principles to be applied within the boundaries imposed by economic, regulatory, and engineering constraints. In fact, the actual chromatographic process used at large scale is dictated equally by these factors as on the effectiveness of the process for purifying the product of interest. [ABSTRACT FROM AUTHOR]

Peptide synthesis has undergone a major transformation in the last three decades, building on the solid-phase synthesis methodology of Bruce Merrifield first published in 1963 (1). During the 1970s, the first automation of peptide synthesis was undertaken using Boc chemistry. In the 1980s, improvements were made in the Boc chemistry automated process and, consequently, the synthesis of more-difficult sequences, as well as longer polypeptides became possible (2). Solid-phase Fmoc synthesis was developed in the early 1980s (3,4) and was also applied to automated systems (5). The 1990s saw improvements in both Boc and Fmoc chemistry together with novel modes of activation of the amino acids in both chemistries (6,7). The result was faster cycle times and, hence, reduced synthesis times. The range of protecting groups and resins available today means that sophisticated syntheses utilizing a combination of Boc and Fmoc chemistry are possible (8). [ABSTRACT FROM AUTHOR]

High-performance liquid chromatography (HPLC) has become the most significant analytical technique of the last 30 years. Whereas HPLC has revolutionized analytical chemistry in general, its significance is mostly recognized in the development of modern biochemistry/biotechnology. Indeed, the scientific accomplishments of many biological disciplines can be largely credited to the development of modern HPLC (1). The popularity and success of the technique has arisen from synergistic improvements in surface chemistry, column technology, instrumentation, and software developments. HPLC is now used for biopolymer purification at the research level, large-scale purifications, and analysis at the development level. In particular, the efficiency, speed, and recovery accomplished by HPLC allowed never seen development of modern biotechnology and pharmaceutical R&D. [ABSTRACT FROM AUTHOR]

Purification of trace compounds (e.g., growth factors, receptors) from bulk biological samples typically requires high purification factors to achieve purification to homogeneity (1-9). The sequential use of microcolumns of varying selectivity, assuming that they have good recovery characteristics, allows very high purification factors to be achieved. As described in Chapter 11, the use of short narrow-bore (2.1 mm id), microbore (1 mm id), or capillary (<1 mm id) columns allows the recovery of purified proteins and peptides in reduced volume at increased concentration compared with larger bore columns. Indeed, as shown in Table 1, it can be calculated that purification factors far in excess of those achieved by two-dimensional polyacrylamide gel electrophoresis can be readily achieved using multidimensional purification protocols, with the added advantage that the purified sample is in a form directly compatible with downstream analysis. Successful micropreparative HPLC requires minimal losses of both mass and biological activity during the chromatographic purification and other associated nonchromatographic sample manipulation (e.g., sample dilution, pH adjustment, storage, chemical manipulation) which we refer to as micromanipulation (3,6,7). By maintaining high overall recovery throughout the procedure, it is possible to take a sample though successive chromatographic steps (i.e., multidimensional purification) and still have sufficient material for structural and/or biological analysis. [ABSTRACT FROM AUTHOR]

The introduction of high-performance liquid chromatography (HPLC) for the analysis and separation of peptides and proteins in the late 1970s offered unrivalled advantages in terms of speed, resolution, sensitivity, and recovery when used for the purification of low microgram levels of complex mixtures of peptides and proteins (1,2). However, the conventional HPLC columns of typically 4.6 mm id and operated at flow rates of 1 mL/min resulted in peak volumes of approx 1 mL or even larger. The resultant sample concentrations (μg/mL) were not ideally suited to subsequent manipulations, and at such low concentrations, losses caused by nonspecific adsorption on either the chromatographic support or associated equipment (e.g., syringes, sample vials, recovery vials) were commonplace (3,4). Attempts to reduce peak volumes by operating at lower flow rates were shown to be associated with poor recoveries of, in particular, hydrophobic proteins (5,6). [ABSTRACT FROM AUTHOR]

Capillary liquid chromatography (capLC) utilizes columns with inner diameters below 0.5 mm and flow rates from some few μL/min down to the μL/min range. Miniaturization of chromatographic procedures using capLC offers substantial advantages over conventional LC separation methods. Capillary columns show increased separation efficiency, minimal solvent consumption, higher peak concentration at the detector, and higher peptide recovery. Several reviews on the state of the art can be found in the literature refs. 1-4. [ABSTRACT FROM AUTHOR]

Mass spectrometry has become one of the preferred methods of detection for high-performance liquid chromatography (HPLC) analyses of biopolymers for a broad range of applications. The primary reason for its widespread use is the value of the information obtained from these types of measurements. When liquid chromatography is interfaced directly to mass spectrometry (LC-MS), molecular weight information can be retrieved from the mass spectra acquired continuously as various components elute from the chromatographic column. In some cases, the identity of the protein or peptide can be assigned immediately if the mass measurements are determined with sufficient accuracy. However, it is more likely that additional information regarding molecular structure is required for identification. In tandem mass spectrometry experiments (LC-MS/MS), structural data are readily generated by fragmentation of peptides (or protein-derived peptides) in the mass spectrometer using the technique of collision-induced dissociation (CID). The fragments generated with CID all originate from the precursor; thus, supplementary information relating to the primary sequence and post-translational modifications of the protein or peptide is obtained. [ABSTRACT FROM AUTHOR]

Immunoaffinity chromatography offers to the chromatographer the exquisite specificity of the antibody's complementarity determining regions (CDRs), or hypervariable regions (1,2). These highly selective loops on the antibody surface capture an antigen with high affinity, while having little interaction with impurities that may also be present. When appropriately immobilized, an antibody retains its affinity for its antigen while being held covalently on the chromatographic support. With appropriate wash conditions, 1000-fold or higher impurity clearances are common. With appropriate elution conditions, 90% or more of the activity present in the chromatographic injection may be recovered in the elution peak. And, quite frequently, it is possible to regenerate the immunoaffinity column to allow its use for 100 or more cycles depending on the impurity levels in the injection. [ABSTRACT FROM AUTHOR]

Immobilized metal ion affinity chromatography (IMAC) of proteins is a high-performance liquid chromatography (HPLC) technique. It has the ability to differentiate a single histidine residue on the surface of a protein (1), it can bind proteins with dissociation constants of 10−5 to 10−7 (2), and has had wide application in the field of molecular biology for the rapid purification of recombinant proteins. [ABSTRACT FROM AUTHOR]

In the past, reverse-phase high-performance liquid chromatography (RP-HPLC), which separates solutes based on hydrophobic interactions, has been established as the most commonly used method for separating peptides and proteins. Its advantages include excellent efficiency and resolving power along with reasonable elution times, and the availability of volatile mobile phases that eliminate the need for a desalting step. The application of this method, however, is limited when a low selectivity or the presence of complex protein mixtures does not permit satisfactory separation of individual components. In such cases, methods are successfully applied that are based on separation mechanisms other than hydrophobic interactions, like electrostatic (seeChapter 3) or hydrophilic interactions or different molecular size (Chapter 5). [ABSTRACT FROM AUTHOR]

Gel filtration chromatography (sometimes referred to as size exclusion chromatography) separates biomolecules based on differences in their molecular size. The process employs a gel media suspended in an aqueous buffer solution which is commonly packed into a chromatographic column. These columns can vary in size from very small (for example, spin columns of <1 mL bed volume for analytical separations) to very large (for preparative scale applications). The gel media consists of spherical porous particles of carefully controlled pore size through which biomolecules diffuse to different extents based on differences in their molecular sizes. Small molecules diffuse freely into the pores and their movement through the column is retarded, whereas large molecules are unable to enter the pores and are therefore eluted earlier. Hence, molecules are separated in order of decreasing molecular weight, with the largest molecules eluting from the column first. [ABSTRACT FROM AUTHOR]

Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of molecules on the basis of hydrophobicity. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase, i.e., the sorbent. A schematic diagram showing the binding of a peptide or a protein to a reversed-phase surface is shown in Fig. 1. The solute mixture is initially applied to the sorbent in the presence of aqueous buffers, and the solutes are eluted by the addition of organic solvent to the mobile phase. Elution can proceed either by isocratic conditions where the concentration of organic solvent is constant, or by gradient elution whereby the amount of organic solvent is increased over a period of time. The solutes are, therefore, eluted in order of increasing molecular hydrophobicity. RP-HPLC is a very powerful technique for the analysis of peptides and proteins because of a number of factors that include: (1) the excellent resolution that can be achieved under a wide range of chromatographic conditions for very closely related molecules as well as structurally quite distinct molecules; (2) the experimental ease with which chromatographic selectivity can be manipulated through changes in mobile phase characteristics; (3) the generally high recoveries and, hence, high productivity; and (4) the excellent reproducibility of repetitive separations carried out over a long period of time, which is caused partly by the stability of the sorbent materials under a wide range of mobile phase conditions (1,2). [ABSTRACT FROM AUTHOR]

High-performance liquid chromatography (HPLC) is now firmly established as the premier technique for the analysis and purification of a wide range of molecules. In particular, HPLC in its various modes has become the central technique in the characterization of peptides and proteins and has, therefore, played a critical role in the rapid advances in the biological and biomedical sciences over the last 10 years. [ABSTRACT FROM AUTHOR]