Two-Dimensional Gel Electrophoresis and 2D-DIGE.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to be one of the most versatile and widely used techniques to study the proteome of a biological system, particularly in the separation of intact proteins. A modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labeling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE gels for differential quantitative analysis of protein expression between experimental groups. Both these methods have distinct advantages in the separation and identification of thousands of individual protein species including protein isoforms and post-translational modifications. This chapter discusses the principles of 2D-PAGE and 2D-DIGE including limitations to the methods. 2D-PAGE and 2D-DIGE continue to be popular methods in bioprocessing-related research, particularly on recombinant Chinese hamster ovary cells, which are also discussed in this chapter.

Top-Down Proteomics and Comparative 2D-DIGE Analysis.

The combination of large-scale protein separation techniques, sophisticated mass spectrometry, and systems bioinformatics has led to the establishment of proteomics as a distinct discipline within the wider field of protein biochemistry. Both discovery proteomics and targeted proteomics are widely used in biological and biomedical research, whereby the analytical approaches can be broadly divided into proteoform-centric top-down proteomics versus peptide-centric bottom-up proteomics. This chapter outlines the scientific value of top-down proteomics and describes how fluorescence two-dimensional difference gel electrophoresis can be combined with the systematic analysis of crucial post-translational modifications. The concept of on-membrane digestion following the electrophoretic transfer of proteins and the usefulness of comparative two-dimensional immunoblotting are discussed.

Comparative Two-Dimensional Fluorescence Gel Electrophoresis.

Two-dimensional comparative fluorescence gel electrophoresis (CoFGE) uses an internal standard to increase the reproducibility of coordinate assignment for protein spots visualized on 2D polyacrylamide gels. This is particularly important for samples that need to be compared without the availability of replicates and thus cannot be studied using differential gel electrophoresis (DIGE). CoFGE corrects for gel-to-gel variability by co-running with the sample proteome a standardized marker grid of 80-100 nodes, which is formed by a set of purified proteins. Differentiating of reference and analyte is possible by the use of two fluorescent dyes. Variations in the y-dimension (molecular weight) are corrected by the marker grid. For the optional control of the x-dimension (pI), azo-dyes can be used. Experiments are possible in both vertical and horizontal (h) electrophoresis devices, but hCoFGE is much easier to perform. The CoFGE experimental principle can additionally be used for protein quantification. For data analysis, commercial software has been adapted.

DIGE-Based Phosphoproteomic Analysis.

Here, we describe a detailed step-by-step protocol for the detection of phosphoproteins in two-dimensional difference gel electrophoresis (2D-DIGE) gels. A standard 2D-DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements 2D-DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples.

DIGE-Based Biomarker Discovery in Blood Cancers.

Cancer of blood or bone marrow-derived cells dysregulates normal hematopoiesis and accounts for over 6% of all cancer cases annually. Proteomic analyses of blood cancers have improved our understanding of disease mechanisms and identified numerous proteins of clinical relevance. For many years, gel-based proteomic studies have aided in the discovery of novel diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, in various diseases, including blood cancer. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) facilitates comparative proteomic research to identify differential protein expression in a simple and reproducible manner. The versatility of 2D-DIGE as a quantitative proteomic technique has provided insight into various aspects of blood cancer pathology, including disease development, prognostic subtypes, and drug resistance. The ability to couple 2D-DIGE with additional downstream mass spectrometry-based techniques yields comprehensive workflows capable of identifying proteins of biological and clinical significance. The application of 2D-DIGE in blood cancer research has significantly contributed to the increasingly important initiative of precision medicine. This chapter will focus on the influential role of 2D-DIGE as a tool in blood cancer research.

DIGE Analysis of ProteoMiner™ Fractionated Serum/Plasma Samples.

The discovery of clinically relevant biomarkers using gel-based proteomics has proven extremely challenging, principally because of the large dynamic range of protein abundances in biofluids such as blood and the fact that only a small number of proteins constitute the vast majority of total blood protein mass. Various separation, depletion, enrichment, and quantitative developments coupled with improvements in gel-based protein quantification technologies, specifically fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), have contributed to significant improvements in the detection and identification of lower abundance proteins. One of these enrichment technologies, ProteoMiner, is the focus of this chapter. The ProteoMiner technology utilizes hexapeptide bead library with huge diversity to bind and enrich low-abundance proteins but at the same time suppresses the concentration of high-abundance proteins in subsequent analysis.

Comparative 3-Sample 2D-DIGE Analysis of Skeletal Muscles.

The skeletal muscle proteome consists of a large number of diverse protein species with a broad and dynamic concentration range. Since mature skeletal muscles are characterized by a distinctive combination of contractile cells with differing physiological and biochemical properties, it is essential to determine specific differences in the protein composition of fast, slow, and hybrid fibers. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a powerful comparative tool to analyze fiber type-specific differences between predominantly fast contracting versus slower twitching muscles. In this chapter, the application of the 2D-DIGE method for the comparative analysis of different subtypes of skeletal muscles is outlined in detail. A standardized proteomic workflow is described, involving sample preparation, protein extraction, differential fluorescence labeling using a 3-CyDye system, first-dimension isoelectric focusing, second-dimension slab gel electrophoresis, 2D-DIGE image analysis, protein digestion, and mass spectrometry.

DIGE Analysis of Clinical Specimens.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2D-GE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The use of an internal pooled standard makes 2D-DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. Technical limitations of this technique (i.e., underrating of low abundant, high molecular mass and integral membrane proteins) are counterbalanced by the incomparable separation power which allows proteoforms and unknown PTM (posttranslational modification) identification. Moreover, the image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

Two-Dye Versus Three-Dye DIGE for Comparative Testis Tissue Proteomic Analysis.

The global analysis of the proteome is an important tool in cell biology. Comparative proteomic evaluations can identify and compare the composition, dynamics, and modifications between different samples. Comparing tissue proteomes under different conditions is crucial for advancing the biomedical field. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a sensitive and robust biochemical method that can compare multiple protein samples over a broad dynamic range on the same analytical gel and can be used to establish differentially expressed protein profiles between different sample groups. 2D-DIGE involves fluorescently labeling protein samples with CyDye flours, via a two-dye or a three-dye system, pre-separation by isoelectric point, and molecular weight. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization, thus enabling accurate high-resolution analysis of differences in protein abundance between samples. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of two-dye and three-dye DIGE minimal labeling.

Two-CyDye-Based 2D-DIGE Analysis of Aged Human Muscle Biopsy Specimens.

The gradual loss of skeletal muscle mass during aging and associated decline in contractile strength can result in reduced fitness, frailty, and loss of independence. In order to better understand the molecular and cellular mechanisms that underlie sarcopenia of old age and the frailty syndrome, as well as identify novel therapeutic targets to treat age-related fiber wasting, it is crucial to develop a comprehensive biomarker signature of muscle aging. Fluorescence two-dimensional gel electrophoresis (2D-DIGE) in combination with sensitive mass spectrometry presents an ideal bioanalytical tool for biomarker discovery in biogerontology. This chapter outlines the application of the 2D-DIGE method for the comparative analysis of human biopsy specimens from middle-aged versus senescent individuals using a two-CyDye-based method.

Identification of Subproteomic Markers for Skeletal Muscle Profiling.

The biochemical and cell biological profiling of contractile fiber types and subcellular structures plays a central role in basic and applied myology. Mass spectrometry-based proteomics presents an ideal approach for the systematic identification of proteomic and subproteomic markers. These representative components of fast versus slow muscle fibers and their subcellular fractions are highly useful for in-depth surveys of skeletal muscle adaptations to physiological challenges, as well as the improvement of diagnostic, prognostic, and therapy-monitoring methodologies in muscle pathology. This chapter outlines the identification of subproteomic markers for skeletal muscle profiling based on bottom-up and top-down approaches, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE).

Sample Preparation and Protein Determination for 2D-DIGE Proteomics.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a widely employed method for efficient protein separation and the determination of abundance changes in distinct proteoforms. This makes this gel-based method a key technique of comparative approaches in top-down proteomics. For the appropriate screening of proteome-wide alterations, initial preparative steps involve sample handling, homogenization, subcellular fractionation, and the determination of protein concentration, which makes the optimal application of these techniques a crucial part of a successful initiation of a new 2D-DIGE-based analysis. This chapter describes sample homogenization and a standardized protein assay for the preparation of homogenates with a known protein concentration for subsequent differential fluorescent tagging and two-dimensional gel electrophoretic separation.

DIGE Analysis of Fish Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) appears to be especially useful in quantitative approaches, allowing the co-separation of proteins of control samples and proteins of treated/disease samples on the same gel, eliminating gel-to-gel variability. The principle of 2D-DIGE is to label proteins prior to isoelectric focusing and use three spectrally resolvable fluorescent dyes, allowing the independent labeling of control and experimental samples. This procedure makes it possible to reduce the number of gels in an experiment, allowing the accurate and reproducible quantification of multiple samples. 2D-DIGE has been found to be an excellent methodical tool in several areas of fish research, including environmental pollution and toxicology, the mechanisms of development and disorders, reproduction, nutrition, evolution, and ecology.

Protein Digestion for 2D-DIGE Analysis.

In-gel digestion of protein spots derived from two-dimensional gels and their subsequent identification by mass spectrometry is involved in a multitude of mass spectrometry-driven proteomic experiments, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). This type of proteomic methodology has been involved in the establishment of comparative proteome maps and in the identification of differentially expressed proteins and their isoforms in health and disease. Most in-gel digestion protocols follow a number of common steps including excision of the protein spots of interest, destaining, reduction and alkylation (for silver-stained gels), and dehydration and overnight digestion with the proteolytic enzyme of choice. While trypsin has been a mainstay of peptide digestion for many years, it does have its shortcomings, particularly related to incomplete peptide digestion, and this has led to a rise in popularity for other proteolytic enzymes either used alone or in combination. This chapter discusses the alternative enzymes available and describes the process of in-gel digestion using the enzyme trypsin.

Subcellular Fractionation for DIGE-Based Proteomics.

Mass spectrometry-based protein methodologies have revolutionized the field of analytical biochemistry and enable the identification of hundreds to thousands of proteins in biological fluids, cell lines, and tissue. This methodology requires the initial separation of a protein constellation, and this has been successfully achieved using gel-based techniques, particularly that of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). However, given the complexity of the proteome, fractionation techniques may be required to optimize the detection of low-abundance proteins, which are often underrepresented but which may represent important players in health and disease. Such subcellular fractionation protocols typically utilize density-gradient centrifugation and have enabled the enrichment of crude microsomes, the cytosol, the plasmalemma, the nuclei, and the mitochondria. In this chapter, we describe the experimental steps involved in the enrichment of crude microsomes from the skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative 2D-DIGE analysis.

DIGE Analysis of Immunodepleted Plasma.

This chapter focuses on upstream immunodepletion of high-abundance proteins from plasma samples and subsequent analysis by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). The abundances of proteins in biofluid proteomes, such as serum, plasma, saliva, and bronchoalveolar lavage fluid (BALF), can exceed ten orders of magnitude. This substantial dynamic range is problematic for the detection of medium and low-abundance proteins by 2D-DIGE analysis. To increase the detection, quantification, and identification of medium-low-abundance proteins, the targeted depletion of known abundant proteins with antibody columns has been successfully employed. From the literature, it is clear that the performance of abundant protein depletion with immunodepletion columns has been successful in broadening the coverage of the biofluid proteome and facilitating the identification of disease-specific biomarkers. The task for a successful biomarker strategy involves the combination of a reproducible and robust fractionation method, coupled with a highly accurate quantitative method, a task that is exemplified by combining both immunodepletion and 2D-DIGE together to discover significant proteins associated with the disease phenotype.

Elucidating Cellular Metabolism and Protein Difference Data from DIGE Proteomics Experiments Using Enzyme Assays.

Assays for measuring enzyme activity can be useful tools for proteomics applications. Enzyme testing can be performed to validate an experimental system prior to a difference gel electrophoresis (DIGE) proteomic experiment and can also be utilized as an integral part of multifaceted experiment in conjunction with DIGE. Data from enzyme tests can be used to corroborate results of DIGE proteomic experiments where an enzyme or enzymes are demonstrated by DIGE to be differentially expressed. Enzyme testing can also be utilized to support data from DIGE experiments that demonstrate metabolic changes in a biological system. The different types of enzyme assays that can be performed in conjunction with DIGE experiments are reviewed alongside a discussion of experimental approaches for designing enzyme assays.

Enzyme Assay Methods to Validate DIGE Proteomics Data.

Enzyme activity assay methods can be used to corroborate the results generated by difference gel electrophoresis (DIGE) proteomic experiments. Two assay methods were chosen to demonstrate how this can be achieved. Assays for determining the activity of superoxide dismutase and NADH dehydrogenase are outlined in detail in this chapter. These methods were chosen as examples because they are frequently used in conjunction with DIGE proteomics.

Immunoblot Analysis of DIGE-Based Proteomics.

Proteins can be separated according to their size by gel electrophoresis and further analyzed by Western blotting. The proteins can be transferred to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF), which results in a replica of the protein's separation patterns. The proteins on the membrane can be detected by specific antibodies followed by visualization either on the membrane itself, on film, or by CCD cameras. Western blotting is a sensitive technique to verify data obtained from fluorescence two-dimensional difference gel electrophoresis (2D-DIGE)-based proteomics.