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Tandem mass spectrometry is a powerful mixture analysis technique suitable for sequence analysis of peptides (1). A tandem mass spectrometer uses two stages of analysis to generate structurally informative fragmentation. The first stage involves separation of an ion from all the other ions that may be entering the mass spectrometer analyser. Ion isolation can be accomplished by separating an ion in time or in space; these processes are used by ion trap instruments or by triple quadmpole instruments, respectively (2-4). The isolated ion is then subjected to ion activation using energetic gas-phase collisions. In the second analysis stage, the mass-to-charge ratio (m/z) values of the fragmentation products are determined. This method is used for protein sequence analysis by first creating a collection of peptides using site-specific enzymatic or chemical proteolysis (1). The collection of peptides is introduced into the mass spectrometer through a separation technique (liquid chromatography [LC] or capillary electrophoresis [CE]) or by batch infusion and finally ionized using electrospray ionization (5-7). Computer control of the data acquisition process allows highly efficient acquisition of these tandem mass spectra as well as unassisted operation of the mass spectrometer (8,9). The resulting tandem mass spectra can reveal the amino acid sequence of peptides by interpretation, or, with the recent expansion of sequence databases, the tandem mass spectra can be used to search protein and nucleotide sequence databases directly to identify the amino acid sequence represented by the spectrum (l,8,10,11). Because tandem mass spectra can be acquired quickly and selectively on individual peptides present, the identities of proteins in a mixture can be determined (10,12,13). [ABSTRACT FROM AUTHOR]

Autoradiography is used to visualize and quantitate radiolabeled proteins that are resolved by 2-D protein gel electrophoresis. Proteins are commonly labeled in vivo with either 3H, 14C, 35S (low-energy (β-emitters), 32P (high-energy β-emitter), or 125I (high-energy γ-rays). During film-based autoradiography, these emitted particles or γ-rays enter the film and cause the ejection of electrons from silver halide crystals generating local precipitates of silver atoms. In the past several years, the use of phosphor imaging has been replacing film-based autoradiography. Phosphor imaging has 10-250 times increased sensitivity compared to film-base autoradiography. While film has a dynamic range of about 300 to 1, phosphor imaging has a linear dynamic range over several orders of magnitude (1). [ABSTRACT FROM AUTHOR]

Compared to classical two-dimensional electrophoresis (2-DE) with carrier ampholytes (1,2), the advent of 2-DE with immobilized pH gradients (IPG-Dalt) (3) has produced significant improvements in 2-D electrophoretic separation with respect to: 1.Higher resolution by using well-defined narrow-pH gradients (3).2.Improved reproducibility, as was demonstrated by interlaboratory comparisons (4,5).3.Higher loading capacity for micropreparative runs, which has accelerated spot identification by microsequencing and mass spectrometry (6-9).4.The possibility to resolve—under steady-state conditions—alkaline proteins, which are normally lost by the cathodic drift of carrier ampholyte focusing or separated by nonequilibrium pH gradient electrophoresis (NEPHGE) with limited reproducibility (10). [ABSTRACT FROM AUTHOR]

Silver staining of polyacrylamide gels was introduced in 1979 by Switzer et al. (1) and rapidly gained popularity owing to its high sensitivity, ca. 100 times higher than staining with Coomassie blue. However, the first silver-staining protocols were not trouble-free. High backgrounds and silver mirrors were frequently experienced, with a subsequent decrease in sensitivity and reproducibility. This led many authors to suggest improved protocols, so that more than 100 different silver-staining protocols for proteins in polyacrylamide gels can be found in the literature. However, all of them are based on the same principle (seerefs. 2 and 3 for details) and comprise more or less four major steps. [ABSTRACT FROM AUTHOR]

The detection of radiolabeled proteins is of fundamental importance in experimental biology. 35S- and 14C-labeled proteins can provide investigators with information about protein expression, synthesis, and degradation. Moreover, posttranslational modifications, such as phosphorylation, can be studied with the use of 32P. Traditional methods for detecting radiolabeled proteins involve somewhat lengthy exposures to photographic film. Quantitation from film-based autoradiography can be achieved by densitometry of repeated and various exposures to films because of the small linear dynamic range of photographic film. [ABSTRACT FROM AUTHOR]

2-D gel electrophoresis is one of the most effective techniques for high-resolution separation of complex protein mixtures. Recent developments in the field of protein mass spectrometry allow the rapid and highly sensitive identification of proteins, and thus amplify the power of preparative 2-D gel electrophoresis. [ABSTRACT FROM AUTHOR]

Shortly after the introduction of electrospray as a viable ionization technique for large molecules (1), electrospray tandem mass spectrometry (ES MS/MS) techniques, such as HPLC-ES MS/MS, were used successfully for peptide sequencing at picomole and subpicomole levels (2-4). In LC-MS/MS, peptide sequence information is generated during the short time, 10-30 s, that a peptide elutes from the HPLC column run at a flow rate of 0.5-5 μL/min. This time frame rarely allows optimization of experimental parameters for MS/MS sequencing of individual peptides unless several LC-MS/MS experiments can be performed on a sample. With the introduction of the nanoelectrospray ion source (5-7), the time constraint of tandem mass spectrometry has been removed, and peptide sequencing has been reliably extended to the femtomole level of gel-isolated protein. [ABSTRACT FROM AUTHOR]

The sequencing of complete genomes is providing an enormous resource for understanding the biology of organisms (1). A complete genome sequence, however, will still leave many unanswered questions about the basic biology of the organism. Which open reading frames are authentic genes? What genes express highly abundant proteins in the cell? How well do the observed physical properties of a protein agree with the predictions of the genomic sequence? Which proteins are covalently processed? Analysis of the proteins encoded in the genome or "the proteome" can answer many of these questions, and will help in understanding the physiology of the organism. [ABSTRACT FROM AUTHOR]

Protein identification and analysis software performs a central role in the investigation of proteins from two-dimensional (2-D) gels. For protein identification, the user matches certain empirically gained information against a protein database to define a protein as already known or as novel. For protein analysis, information in protein databases can be used to predict certain properties about a protein, which can be useful for its empirical investigation. The two processes are thus complementary. Although there are numerable programs available for the above-mentioned applications, we have developed a set of original tools with a few main goals in mind. Specifically, these are: 1.To utilize the extensive annotation available in the SWISS-PROT database (1) wherever possible.2.To have tools specifically, but not exclusively, applicable to proteins prepared by 2-D gel electrophoresis.3.To have all tools available on the World Wide Web and freely available to the scientific community. [ABSTRACT FROM AUTHOR]

Polyacrylamide gel electrophoresis (PAGE) is probably the most general and widespread protein separation technique. This simple method is used in almost every molecular biology and biochemistry laboratory to monitor protein experiments, including consecutive stages of protein purification. PAGE may even be the final step of a purification protocol. Over the last decade, two-dimensional gel electrophoresis (2-D-PAGE) (1) has been developed, and applied to the separation of crude protein mixtures from organelles, tissues, and whole organisms (2). [ABSTRACT FROM AUTHOR]

Methods for the identification of proteins have advanced dramatically this decade through the introduction of mass spectrometric techniques and instrumentation sensitive enough to be applicable to biological systems (1). The two mass spectrometric techniques that have provided these advantages are electrospray-ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) (seeChapters 52, 54, and 55). [ABSTRACT FROM AUTHOR]

Methods combining the high throughput and sensitivity of matrix-assisted laser desorption ionization mass spectometry (MALDI-MS) with polyacrylamide gel electrophoresis (PAGE) are rapidly gaining attention, but almost exclusively for applications eluting proteolytic digest products from gels and membranes (1-9). These methods fulfill a considerable need for identifying unknown proteins isolated in polyacrylamide gels, but they do not provide masses for intact proteins, a particular concern should the goal be the understanding of anomalous electrophoretic migrations, characterizing the complete ensemble of posttranslational modifications displayed by proteins, or understanding why multiple protein spots on a two-dimensional (2-D) gel are all identified as the same protein. These issues are particularly relevant for low-abundance proteins, which may be identified by matching the masses of only a few tryptic peptides to those predicted from cleavage of an unmodified protein, or by matching small stretches of sequence information (9,10). In some cases, those matches may correspond to <10% of the total protein sequence (9). Analysis of intact proteins has been performed following electroblotting to membranes (11-19), but we will describe a method to link gel electrophoresis directly to MALDI-MS, yielding masses of both intact proteins and cleavage products without electroelution or electroblotting (20-22). Key to the success of this approach are ultrathin polyacrylamide gels, which dry to thicknesses of 10 μm or less and which have the additional advantages of rapid preparation and electrophoresis run times. [ABSTRACT FROM AUTHOR]

For years, proteolytic digest and peptide purification were essential tools for the biochemist wishing to determine the primary structure of a protein. The strategies changed with the availability of a polypeptide gas-phase sequencer and the advent of DNA recombinant technology. However, blocked N-termini and changing trends in molecular cloning techniques, such as PCR, brought back "internal" sequencing as well, a term coined by Aebersold et al. in their seminal paper on the in situ micropreparative digestion of electroblotted proteins (1). Since then, many practical improvements of the original digest recipe and alternative approaches have been proposed (2). Considerable effort has also been expended to interface in situ digestion with micro-liquid chromotography (LC), peptide sequencers, and mass spectrometers. The ability to generate a set of specific peptides (e.g., tryptic), together with recent advances in biopolymer mass spectrometry provide a novel approach to protein identification. Accurate masses of several protein fragments compose a "peptide mass fingerprint," theoretically sufficient for unambiguous searching of sequence repositories (3). It is therefore expected that enzymatic proteolysis will become even more widely used in the future. [ABSTRACT FROM AUTHOR]

Two-dimensional electrophoresis (2-DE) is one of the most advanced techniques for direct separation of considerable numbers of proteins. Direct microsequence analysis of the amino (N)-terminal partial sequence of the electroblotted protein spots from the 2-DE gels and the homology search against protein sequence database(s) have provided identification of proteins in addition to the conventional identification processes, such as comigration. [ABSTRACT FROM AUTHOR]

Amino acid analysis is a powerful and sensitive technique for the determination of the amino acid composition of proteins. In the past, the automation of amino acid analysis was based on postcolumn ninhydrin analysis, e.g., the Beckman 6300. However, a demand for high sensitivity and high sample throughput has led to the development of precolumn derivatization of amino acids and subsequent separation by reverse phase-high performance liquid chromatography RP-(HPLC), e.g., GBC AMINOMATE system. Although the chromophoric or fluorogenic derivatives allow greater sensitivity (100 fmol to low pmol range of protein), protein hydrolysis remains the limiting factor to high sensitive amino acid analysis. At the 5- to 20-pmol level, various contaminants influence the accuracy of both the qualitative and quantitative aspects of the analysis. Therefore, correct procedure and careful manipulation are required to achieve a successful analysis. [ABSTRACT FROM AUTHOR]

First devised in a time dominated by one-dimensional SDS-PAGE, immunoblotting (also dubbed "Western" blotting, [1,2]), is now widely used in conjunction with 2-D PAGE (electrofocusing/SDS-PAGE), whose diffusion is favored by its long-awaited standardization (3,4) and by the coming of age of proteome science and technology (5,6). [ABSTRACT FROM AUTHOR]

Bacterial genomes range greatly in size, encoding from as few as 500 to more than 4000 proteins. The tools of bioinformatics have become more and more accurate at predicting open reading frames within stretches of DNA, and within this decade, the proteome complement of one or more genomes should be completed. To accomplish such a task, methods for detecting and building an inventory of the protein products encoded by the genome must be used. Such methods have been under way for over 20 years, even before the projects to complete the DNA sequence of the bacterial genome were initiated. Using two-dimensional polyacrylamide gel electrophoresis (2-D gels) (1), one can easily detect the proteins bacteria expressed at levels >50 mol/cell. For the bacterium Escherichia coli, about 1200 proteins can readily be detected under any one growth condition. [ABSTRACT FROM AUTHOR]

Proteome analysis has been revolutionized by the marriage of two-dimensional (2-D) gel electrophoresis and mass spectrometry (1-4). Mass spectrometry in combination with database searching permits the large-scale identification of proteins, and 2-D gel electrophoresis allows for the large-scale visualization and separation of cellular proteins. Previously, spot quantitation has been difficult, and numbers obtained were usually comparative, but not absolute (5-7). Mass spectrometry and 2-D gel electrophoresis in tandem can be used to quantitate protein spots. This chapter covers protocols to quantitate precisely the relative amounts of protein observed in the 2-D gels using radiolabeled total cell extracts. [ABSTRACT FROM AUTHOR]

A federated two-dimensional (2-D) database is a database containing 2-D data that are accessible on the World Wide Web (WWW) and that are dynamically linked to other similar databases through active hypertext links over the Internet (1,2). Federated 2-D databases respect five rules that can be found at URL: http://www.expasy.ch/ch2d/fed-rules.html. [ABSTRACT FROM AUTHOR]

In ref. 1, we described a computer-assisted visual method, Flicker, for comparing two two-dimensional (2-D) protein gel images across the Internet is described (see Note 5). This approach may be useful for comparing similar samples created in different laboratories to help putatively identify or suggest protein spot identification. 2-D gels and associated databases are increasingly appearing on the Internet (2-13) in World-Wide Web (WWW or web) (14) servers and through federated databases (15). Table 1 lists some web URL addresses for a number of 2-D protein gel databases that contain 2-D gelimages with many identified proteins. This opens up the possibility of comparing one's own experimental 2-D gel image data with gel images of similar biological material from remote Internet databases in other laboratories. This new data analysis method allows scientists to collaborate and compare gelimage data over the World Wide Web more easily. [ABSTRACT FROM AUTHOR]

Many laboratories have identified proteins on two-dimensional (2-D) maps, and have built a master gel and a related 2-D database containing data specific to the identified proteins. Several of these databases have been made available on the World Wide Web (WWW) (1), and many more will undoubtedly follow. This chapter shows how to retrieve data remotely from these databases over the Internet. [ABSTRACT FROM AUTHOR]

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) images, when used to analyze protein expression in large sets of biological samples, are best processed by computer. In this manner, one can detect and quantify even the faintest spots, quantitatively compare 2-D images among each other, or identify protein expression changes across various sets of 2-D images, such as disease vs control, or different stages in a toxicological study (1). [ABSTRACT FROM AUTHOR]

Prevalent methods for visualizing proteins resolved by two-dimensional (2-D) gel electrophoresis include autoradiography, silver staining, and Coomassie brilliant blue staining (1,2. The organic dye Coomassie brilliant blue is capable of detecting as little as 100 ng of protein, but this is considerably less sensitive than silver staining or autoradiography (1,2. Silver staining allows detection of low-nanogram amounts of protein, but detection sensitivity of autoradiography can be considerably better, since it depends principally on the specific activity of the radiolabeling method. Other staining techniques are valuable for specific applications in 2-D gel electrophoresis, such as Edman-based protein sequencing, mass spectrometry, and immunoblotting. Many of these applications require stains that do not covalently modify proteins, are relatively sensitive, and are easily reversible (3). Additionally, immunoblotting protocols are often based upon the enzymatic formation of a colored formazan product or the chemiluminescent production of light (4). Thus, image acquisition requires detection of diverse chromogenic, radioactive, and luminescent signals. [ABSTRACT FROM AUTHOR]

The profile of the two-dimensional (2-D) separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious "trains" of spots that differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from a variety of posttranslational modifications. There is growing evidence that alterations to the glycosylation of a protein can be correlated with developmental and pathological changes; these changes can be visualized on the 2-D separation by alterations in the pattern of these glycoprotein isoforms. [ABSTRACT FROM AUTHOR]

Electrophoretically separated proteins may be visualized using organic dyes, such as Ponceau red, Amido black, fast green, or most commonly Coomassie Brilliant Blue (1,2). Alternatively, sensitive detection methods have been devised using metal ions and colloids of gold, silver, copper, carbon, or iron (3-12). Metal chelates form a third class of stains, consisting of organometallic complexes that bind avidly to proteins resolved in polyacrylamide gels or immobilized on solid-phase supports (13). The metal chelate staining procedures are simple, requiring reagents that are easily prepared, are stable at room temperature, and can be reused several times without loss of sensitivity. The staining procedures are relatively inexpensive, since they do not utilize precious metals, such as gold or silver. Like Ponceau red stain, the metal chelate stains are readily reversible. Complexes form at acidic pH and elute on increasing the pH to 7.0-10.0. Metal chelates can be used to detect proteins on nitrocellulose, PVDF, and nylon membranes as well as in polyacrylamide gels. The metal complexes do not modify proteins, and are compatible with immunoblotting, lectin blotting, mass spectrometry, and Edman-based protein sequencing (13-17). Metal chelate stains are suitable for routine protein measurement in solid-phase assays owing to the quantitative stoichiometry of complex formation with proteins and peptides (15,16). Such solid-phase protein assays are more sensitive and resistant to chemical interference than their solution-based counterparts (15). [ABSTRACT FROM AUTHOR]

The ability of two-dimensional electrophoresis (2-DE), based on the original method developed more than 20 years ago (1), to separate simultaneously up to several thousand proteins using large-format gels (2) has made it the method of choice for the analysis of protein expression in complex biological systems, such as whole cells, tissues, and organisms. Initially, 2-DE only yielded information on the charge (pI), size (Mr), and relative abundance of the separated proteins. However, in recent years, a variety of methods have been developed that make it possible to identify and characterize proteins separated by 2-DE. Many of these methods depend on the technique of Western electroblotting in which proteins separated by 2-DE are transferred ("blotted") by the application of an electric field perpendicular to the plane of the gel onto the surface of an inert membrane, such as nitrocellulose (3). Methods for electroblotting of protein from 2-DE gels are described in Chapter 35. [ABSTRACT FROM AUTHOR]

The ability of two-dimensional electrophoresis (2-DE) to separate complex mixtures of proteins, such as represented by cells, tissues, and even whole organisms, has been recognized for more than 20 yr (1). Using "standard"-format (around 20×20 cm) 2-D gels, the method is capable of routinely separating 2000 proteins from whole-cell and tissue extracts. The resolution capacity can be extended significantly (up to 5000-10,000 proteins) using large-format (40×30 cm) 2-D gels (2). Over the last 20 yr, 2-DE has been used primarily as an analytical tool for the characterization of proteins by their charge (pI), size (Mr), and relative abundance. Specialized computer software has been developed for the qualitative and quantitative analysis of 2-DE protein patterns (3), and these systems have been used to construct several comprehensive databases of protein expression in a variety of cell types and tissues (for examples, seeref. 4). [ABSTRACT FROM AUTHOR]

The identification of proteins using preparative gel electrophoresis and mass spectrometry requires reversible staining of relatively thick (1-1.5 mm) polyacrylamide gels. We have found that staining with colloidal Coomassie brilliant blue G-250 or negative staining with imidazole-zinc yields high-resolution stains (Fig. 1) that are compatible with subsequent mass spectrometric analysis (see Notes 1 and 5). [ABSTRACT FROM AUTHOR]

A major mechanism that cells use to regulate protein function is by phosphorylation and/or dephosphorylation of serine, threonine, and tyrosine residues. Phosphopeptide mapping of these phosphorylated residues allows investigation into the positive and negative regulatory roles these sites may play in vivo. In addition, phosphopeptide mapping can uncover the specific phosphorylated residue and, hence, kinase recognition sites, thus helping in the identification of the relevant kinase(s) and/or phosphatase(s). [ABSTRACT FROM AUTHOR]

Internal standards are extremely valuable for determining the pH and mol-wt ranges of a specific 2-D gel system and for constructing calibration curves for calculating the relative pI and Mr of unknown proteins. Although external markers are often used for initially calibrating a 2-D gel, eventually the predicted isoelectric points and molecular weights of identified proteins function as calibration markers. For the beginner, several commercial kits are available containing preformulated mixtures of proteins that can be run on 2-D gels either in parallel or mixed with experimental samples as standards. For carrier ampholyte IEF gels, the pH profile can be directly determined by measuring the pH throughout the gel using a standard pH meter. For both CA and IPG IEF gels, a train of differentially carbamylated proteins producing a uniform series of spots on the 2-D gel provides an internal standard for both evaluating the performance of the isoelectric focusing gel and comparing the 2-D spots positions on different gels. However, the exact pI of each spot in the "carbamylated train" is unknown. For calibrating the SDS-PAGE gel, a mixture of protein standards of known molecular weight is simply loaded onto the SDS-PAGE gel flanking both sides of the IEF gel. However, it is important to load the mol-wt standards in the same manner as the IEF gel to minimize any differences in the relative mobilities of the standards and experimental proteins. [ABSTRACT FROM AUTHOR]

The task of separating complex mixtures of proteins into individual components was revolutionized by the development of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Modifications of this technique employing gradient gels significantly improved the resolution of proteins and polypeptides based on their molecular weights. Two-dimensional (2-D) gels that combine initial isoelectric focusing in a tube gel with SDS page electrophoresis on a slab gel produce extremely high resolution of proteins. In this method, each protein migrates to a position determined both by its isoelectric point and molecular weight. If the mixture of proteins is complex, however, it can still be difficult to separate all proteins such that they occupy unique positions on the gel, because many proteins have similar isoelectric points and molecular weights. [ABSTRACT FROM AUTHOR]

Several approaches can be used for identifying 2-DE resolved proteins, including microsequencing, immunoblotting with highly specific antibodies, and mass spectrometry-based technologies (for a review of the latter approaches, seeref. 1). In the case of immunoblotting, although this technique has the advantage of being both fast and specific, it is not always straightforward, since many antibodies fail to recognize proteins following acrylamide gel electrophoresis. This is owing to the fact that some monoclonal antibodies (MAbs) recognize only conformational epitopes, which are dependent on the native spatial conformation of the protein, as distinct from sequential determinants, which are dependent on the linear amino acid sequence of a peptide (for a review, seeref. 2). In contrast to sequential determinants, conformational determinants are frequently destroyed by reagents employed in electrophoresis (e.g., reducing reagents, SDS, and/or urea) (3,4). Although electrophoretic methods used for resolving native proteins have been described (5), these techniques are not widely used because of technical problems, such as protein aggregation and the wide variance in the effective electrophoretic mobility of proteins in different buffer systems. Moreover, native 2-DE in our hands has proven to be time-consuming compared to conventional reducing 2-DE (4). [ABSTRACT FROM AUTHOR]

The second dimension of 2-D-PAGE uses denaturation and detergent, usually sodium dodecyl sulfate (SDS), to separate proteins by exploiting their molecular mass differences. During SDS-PAGE, all the proteins in the mixture have the same net charge per gram, and movement through the gel is based solely on the molecular mass of the proteins. This is ideal for 2-D-PAGE, because the first-dimension isoelectric focusing is charge-based, and thus, the proteins have been separated by two independent parameters. To achieve a separation based on molecular weight, it is necessary to overcome the intrinsic charges of the protein by loading the protein with the charge modifier SDS, an anionic surfactant with a C12 alkyl tail and an ionic head group. SDS swamps the intrinsic charge of proteins, thus disrupting hydrogen bonds and hydrophobic interactions and preventing protein aggregation. When SDS is used in combination with a reducing agent to cleave disulfide bonds, the proteins are unfolded and form ellipsoid shapes coated in SDS. During electrophoresis in a sieving gel, the denatured SDS polypeptides migrate according to molecular size, and there is a linear relationship between the logarithm of the molecular weight and the relative distance the polypeptides move. [ABSTRACT FROM AUTHOR]

After IEF-IPG (seeChapters 21-24), the IPG gel strips are equilibrated in the presence of SDS, DTT, urea, glycerol, and iodoacetamide, and placed onto the surface of a horizontal or on top of a vertical SDS gel (1-3) (seeChapter 26). For horizontal setups, the laboratory-made or ready-made SDS-PAGE gel (ExcelGel SDS), cast on plastic backing, is placed onto the cooling plate of a horizontal electrophoresis system. The equilibrated IPG gel strip(s) is (are) transferred gel-side down onto the surface of the SDS gel alongside the cathodic electrode wick. For vertical setups, the equilibrated IPG gel strips are loaded on top of vertical SDS polyacrylamide gels and embedded in agarose (seeChapter 26). On completion of electrophoresis, the resolved polypeptides are either stained with Coomassie blue or silver nitrate, or are transferred onto an immobilizing membrane and detected with specific reagents, such as lectins or antibodies. Alternatively, they can be subjected to amino acid (sequence) analysis or mass spectrometry (4-8). [ABSTRACT FROM AUTHOR]

The reproducibility and resolving power of immobilized pH gradient (IPG) led two-dimensional gel electrophoresis (2-DE) technology to the heart of proteome projects. Despite the recent progress achieved in this field, 2-DE is still not widely used as a screening tool either in industry or in clinical chemistry laboratories. This is mostly owing to the fact that this methodology is complex, expensive, and slow. Actually, the whole process for a standard 2-DE (160×180 mm) requires three to four working days depending on which procedure is used. In order to reduce significantly the above limiting factors, a mini-2-DE method was developed that has the ability to produce several protein maps easily within one working day (<6 h) at a much lower price. It uses a combination of in-gel sample rehydration (1,2), small nonlinear 3.5-10 IPG gels (7 cm), homemade or precast vertical slab gels, and an automated protein-staining device. Examples of plasma and Escherichia coli protein separation are presented in this chapter. It demonstrates that this method is rapid, simple to perform, and keeps the advantage of the 2-D resolving power. Therefore, it opens the prospect of high 2-DE throughput for sample screening. [ABSTRACT FROM AUTHOR]

Two-dimensional gel electrophoresis (2-DE) is a useful proteomic tool to unravel some of organism gene expression complexities, and to study directly the gene products and several of their corresponding posttranslational modifications. A simple and universal sample application method was developed (1,2) to provide reproducible separation of thousands of polypeptides on a single gel and their transfer to PVDF membrane. This methodology is suitable for commercially available or homemade IPG strips. Practically, we combined "volume-controlled rehydration" and "in-gel sample rehydration," using the entire IPG gel for sample application, with the protein entering the gel during its rehydration. This method avoids the use of sample cups, eliminates precipitation at the sample application point, and thus, improves resolution throughout the pH range of the gel. It also allows precise control of protein amounts and sample volumes loaded into the IPG gels. However, the separation of low-copy-number proteins in amounts sufficient for postseparation analysis continues to present a challenge for 2-D techniques, and prefractionation techniques are still required in this situation. [ABSTRACT FROM AUTHOR]

Two-dimensional electrophoresis (2-DE) is potentially the most powerful technique known for resolving complex protein mixtures. Proteins can be purified to homogeneity in one step in most cases. The applications for 2-DE are numerous and diverse. They include the analysis of protein patterns in various organisms and tissues, monitoring of the purity of fractions derived from chro-matography (or other separation methods), and the determination of the characteristics of a particular protein, such as isoelectric point, molecular weight of subunits, and number of isoforms. [ABSTRACT FROM AUTHOR]

Accurate quantitative and qualitative comparison of resolved proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) requires that identical amounts of sample be loaded on each gel. Use of trichloroacetic acid (TCA) or organic solvent precipitation protocols followed by a protein assay results in either over- or underestimation of solubilized protein concentration. Furthermore, the practice of loading equivalent amounts of radioactively labeled protein-based counts/min or loading extract from the same number of cells is inaccurate. The author has observed that radioactively labeled cell extracts exhibit 10-20% variances in protein concentration. Similarly, solubilization of 2×106 cells from a number of colorectal carcinoma cell lines resulted in protein concentrations ranging from 1.60 to 3.93 μg protein/μL buffer (1). Therefore, the ability to quantify protein actually solubilized in 2-D PAGE sample buffers is necessary to allow users to adjust for variable solubility properties of protein(s). [ABSTRACT FROM AUTHOR]

Immobilized pH †*adients (IPGs) for isoelectric focusing (IEF) were introduced in 1982 (1). After experiencing initial problems in handling IPGs, a basic protocol of two-dimensional electrophoresis (2-DE) with IPGs in the first dimension, followed by horizontal or vertical SDS electrophoresis (IPG-Dalt), was established in 1988 (2). Since that time, the protocol has not been changed essentially. Compared to classical 2-DE with carrier ampholytes (3,4), the employment of IPG-Dalt has produced significant improvements in 2-D electrophoretic separation, permitting higher resolution and reproducibility, which was also demonstrated by an interlaboratory comparison (5). Alkaline proteins, normally lost by the cathodic drift of carrier ampholyte focusing or separated by NEPHGE (6) with limited reproducibility, were perfectly separated under equilibrium conditions (7). These features together with the high loading capacity of IPG-Dalt for micropreparative runs (8-10) have accelerated spot identification by microsequencing, amino acid composition analysis, and mass spectrometry. [ABSTRACT FROM AUTHOR]

One of the main requirements for a 2-D protocol is reproducibility of spot position, and, indeed, the technique of isoelectric focusing on immobilized pH gradients (IPGs) is ideally suited to provide highly reproducible 1-d separations. IPGs are obtained through the copolymerization of acidic and basic acrylamido derivatives of different pKs within a polyacrylamide matrix (1,2) (Fig. 1). The pH gradient may be devised by computer modeling either with a linear or with an exponential course. IPGs are cast from two limiting solutions containing the buffering chemicals at concentrations adjusted to give the required pH course upon linear mixing. For consistent results, gradient pouring and polymerization are carried out under controlled conditions. The covalent nature of the chemical bonds formed during the polymerization step results in a permanent stability of the pH gradient within the matrix. Conflicting requirements during the focusing procedure prevent any effective use of IPGs into capillary tubes (3): The need to buffer with carrier ampholytes (CAs) the pH extremes caused by the migration of the polymerization catalysts is contrasted by the adverse effects of the electroendosmotic flow brought about by the addition of CAs to the gel phase. The demand for the IPG gels to be backed by a binding support—they are usually cast on GelBond™ foils—results in dimensional stability between 1-d and 2-d as a further assistance to reproducibility. [ABSTRACT FROM AUTHOR]

Two-dimensional electrophoresis (2-DE) of proteins is used for several purposes, such as resolving a distinct group of proteins (e.g., serum proteins), revealing the heterogeneity of a particular protein (e.g., actin, transferrin), purifying a protein, or testing the purity of a protein gained by other methods. However, the most exceptional feature of this method is its potentiality to resolve all the various proteins of a certain cell type or tissue. It was this particular feature of the 2-DE technique developed in 1975 (1,2) that opened the way to study many of the biological problems of present interest under a new aspect: Problems of gene expression, gene regulation, genetic variation, cell differentiation, embryonic development, pathogenesis of certain diseases, and other fields could be studied on the basis of a broad spectrum and a representative number of proteins, whereas previously studies like these were performed on selected proteins, often selected because of their easy accessibility and considered as model proteins (e.g. hemoglobin). Moreover, when simple procedures for extracting cell or tissue proteins were used, i.e., procedures that avoid steps, such as protein precipitation, lyophilization, dialyses, or chromatographic fractionations, 2-DE protein patterns revealed the individual proteins in quantities that reflect, at least to some extent, the relative concentration of these proteins in the cells or tissues. Quantitative data on gene expression offer an important parameter for all studies in cell biology. [ABSTRACT FROM AUTHOR]

Several formats—small, standard, and large—can be employed to separate proteins by two-dimensional (2-D) protein electrophoresis. The number of proteins in the sample as well as the degree of resolution needed determine the particular format used. The small format is preferred when few proteins (usually <30 proteins that differ to a moderate extent in size and charge) are to be separated. This format allows for a significantly shortened analysis time. The small gels also are easier to handle than larger ones. Large formats are useful for separating large numbers of proteins (usually >200), many of which may have similar molecular sizes or charges. Large formats provide the highest degree of separating power of all the formats, although some resolution with respect to spot intensity may be lost in some areas of the gel. The standard format is the most commonly employed format, because it provides a significantly higher degree of separating power than the small format and improved spot intensity over the large format. The gels also are much easier to handle than the more cumbersome and fragile large format gels. [ABSTRACT FROM AUTHOR]

Cells, tissues, and organs require energy to function normally. Some of them are metabolic providers, and some others are users. Body fluids act as transporters and distributors of metabolic "fuels" between cells and maintain relatively constant conditions through homeostasis. The ratio of solute to water in all body fluids is maintained within a very narrow range. The concentrations of body fluid metabolites are thus extremely well regulated. Many diseases or environment disturbances lead to changes in the expression of body fluid proteins. Information on these changes is often of diagnostic, prognostic, or therapeutic importance. [ABSTRACT FROM AUTHOR]

The protocol for extracting proteins from mouse and human tissues (organs) described in this chapter follows a strategy that is based on the intention to include all the various protein species of a particular tissue in a set of samples that are suitable for two-dimensional electrophoresis (2-DE), particularly for the large gel 2-DE technique described in Chapter 18. The aim then is the resolution and visualization of all these protein species in 2-DE gels. This aim explains some features of our tissue extraction procedure for gaining the proteins. Fractionation of total tissue proteins was preferred to a one-step extraction of all proteins. Using the fractionation procedure, the many different protein species of a tissue can be distributed over several 2-DE gels, and this increases resolution. However, a postulate is that fractionation of tissue proteins results in fraction-specific proteins. To achieve this, cell fractionation is usually performed with the aim of isolating special cell organelles (nuclei, mitochondria) or cell structures (membranes). The proteins are then extracted from these natural fractions. This procedure, however, includes washing steps to purify the cell fractions, and the elimination of cell components that are not of interest and cell residues, which are rejected. Using this procedure, an uncontrolled loss of proteins is unavoidable. [ABSTRACT FROM AUTHOR]

Differential detergent fractionation (DDF) represents an alternative method for cell fractionation that employs sequential extraction of cells or tissues with detergent-containing buffers to partition cellular proteins into structurally and functionally intact and distinct compartments (1-5). Relative to cell fractionation by differential pelleting, DDF has the advantage of preserving the integrity of microfilament and intermediate filament cytoskeletal networks, and is especially applicable to use with limited quantities of biomaterial (4-6). In addition, DDF is simple, highly reproducible, labor-sparing, and ultracentrifuge-independent. DDF is appropriate for a variety of investigations (7-15), including those objecting to: 1.Enhance the delectability of low-abundance species or semipurify components of known subcellular localization.2.Define the subcellular localization of enzymes, regulatory, or structural proteins as well as nonprotein metabolites.3.Monitor physiologic fluxes and compartmental redistribution of biomolecules under basal and stimulated conditions.4.Identify cytoskeletal-associated and interacting proteins.5.Investigate the role of cytoskeletal networks in the subcellular localization of endogenous and exogenous factors, including mRNA, viral components, and heat shock proteins, interactions relevant to understanding mechanisms of infection, protein turnover, and the stress response. [ABSTRACT FROM AUTHOR]

Two-dimensional polyacrylamide gel electrophoresis (2-DE) provides the ability to resolve and quantify thousands of proteins from an unfractionated eukaryotic cell lysate. The use of 2-DE in studying eukaryotic cells has run the gambit from ozone-stressed Norway spruce needles (1), mapping proteins in the differentiation of mouse endoderm, mesoderm, and ectoderm (2) to the study of apoptosis (3). [ABSTRACT FROM AUTHOR]

The small nematode Caenorhabditis elegans (C. elegans, 1 mm in length) originally was introduced into the laboratory in 1965 by Brenner (1) and has since become an important model organism. Caenorhabditis elegans for a variety of reasons offers excellent conditions for the study of basic features of life (2). C. elegans is surrounded by a rigid cuticula, which is, however, completely transparent. The adult organism is composed of a constant number of somatic cells (959 in the adult hermaphrodite, 1031 in the adult male), of which the complete cell lineage during development is known in all details (3). Altogether, this makes C. elegans a very suitable model organism to approach fundamental problems in development and aging. [ABSTRACT FROM AUTHOR]

Although the majority of proteins expressed in organisms with relatively low protein complexity, such as Escherichia coli, can be resolved and detected in a single gel (1,2), this is not true for more complex organisms, such as Drosophila melanogaster. A smaller fraction of the total complement of proteins can be detected in a single gel as the protein complexity of an organism increases. With current detection techniques, this situation can be improved by analyzing subfractions separately and then matching them to each other, preferably using computer-assisted, gel-matching techniques. It has been estimated that about 8% of the proteins encoded by the genome can be analyzed in a single 2-D PAGE of a total protein extract of D. melanogaster (3). By matching subfractions, that value can be increased dramatically. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae became the first eukaryotic organism to have its entire genome sequenced (1). With the completion of the genome, over 6000 genes on 16 chromosomes were identified. Several laboratories are undertaking the task of identifying the yeast proteins on two-dimensional (2-D) gels with the goal of developing a yeast protein database for studying global changes in protein synthesis, protein modification, and protein degradation that result from environmental or genetic changes (2,3). This chapter provides protocols for labeling yeast with 35S-methionine and the preparation of 2-D extract using methods developed by Garrels et al. and Boucherie et al. (2,3). Additional protocols for preparing yeast 2-D extracts can be found at the Geneva University Hospital's Electrophoresis Laboratory, which can be accessed via the World Wide Web at http://expasy.hcuge.ch/ch2d/technical-info.html and at the University of Göteberg's Lundberg Laboratory, which can be accessed at http://yeast-2dpage.gmm.gu.se/sacch/immobiline/methods/labelling. [ABSTRACT FROM AUTHOR]