Chapter 2 Comparative DIGE Proteomics Kay Ohlendieck Abstract Gel-based proteomics has been widely used for the systematic cataloging of the protein constituents of defined biofluids, purified organelles, individual cell types, heterogeneous tissues and isolated organs, as well as being applied to comparative biochemical and biomedical analyses of complex biological specimens. Of the many electrophoretic techniques used in modern biochemical approaches, large-scale protein separation by difference gel electrophoresis (DIGE) has established itself as the most powerful analytical tool in comparative proteomics. Both 2-dye and 3-dye fluorescence systems with minimal or saturation labeling are routinely used. This chapter briefly describes the technical advantages of the pre-electrophoretic fluorescent labeling technique and discusses the bioanalytical usefulness of this highly successful electrophoretic method. Key words CyDye, Difference gel electrophoresis, Difference in-gel electrophoresis, DIGE, Fluorescence labeling, Gel electrophoresis, Mass spectrometry, Proteomics, Two-dimensional gel electrophoresis
1
Introduction The successful isolation, identification, and detailed characterization of individual protein species, complex protein assemblies, organellar subproteomes, or entire proteomes by analytical biochemistry rely heavily on the appropriate combination of a variety of standardized techniques. The routine workflow in many protein biochemical and proteomic studies includes: (1) the efficient extraction of proteins with widely differing physicochemical properties from complex biological samples, (2) the careful protection of sensitive proteins from degradation during lengthy isolation procedures, (3) the basic separation of structurally or functionally related protein populations by relatively crude subcellular fractionation methods such as differential or density gradient centrifugation, (4) the refined separation of different protein classes by the systematic application of large-scale methods such as gel
Kay Ohlendieck (ed.), Difference Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1664, DOI 10.1007/978-1-4939-7268-5_2, © Springer Science+Business Media LLC 2018
17
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electrophoresis, liquid chromatography, and/or affinity methods, (5) the reliable identification of representative peptides, individual protein fragments or intact proteins by mass spectrometry or antibody-based techniques, (6) the swift determination of protein concentration and isoform expression patterns, and (7) the comprehensive determination of post-translational modifications [1–3]. Over the last few decades of bioresearch, gel electrophoresis has played a central role in both preparative and analytical terms [4] and has been widely employed for efficient peptide and protein separation in the more recently established field of mass spectrometry-based proteomics [5–7]. Although a variety of liquid chromatographic methods are now frequently incorporated in proteomic studies, gel electrophoretic techniques are at the core of modern proteomics and routinely employed in the systems biological analysis of proteome-wide changes and adaptations in health and disease [8–10]. Many different electrophoretic approaches are employed in analytical biochemistry, whereby difference gel electrophoresis (DIGE) presents one of the most powerful comparative techniques in modern proteomics [11]. Using particular DIGE-based methods, the efficient gel electrophoretic separation of proteins is mostly based on natural or modified differences in overall charge between individual polypeptide chains, as well as dissimilarities in molecular size under native or denatured conditions. This chapter briefly outlines the development, technical advances, and applications of DIGE in modern proteomics.
2
Gel-Based Proteomics and DIGE Analysis In general, gel electrophoretic techniques can be distinguished based on the dimensionality of the gel system and the main labeling procedure for the visualization of protein bands or spot patterns. Routinely used one-dimensional gel electrophoretic methods for the separation of proteins include isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis [4]. A combination of both techniques in two-dimensional gel electrophoresis is significantly more efficient for large-scale protein separation approaches due to its huge capacity and high resolution, making it a frequently used method in high-throughput proteomic surveys [8]. Depending on the particular gel electrophoretic technique and the analyzed samples, several hundred to thousands of individual protein spots can be visualized by standardized two-dimensional gel electrophoresis [5]. Besides optimizing the electrophoretic procedure, protein labeling is a crucial step in gel-based analytical proteomics. Standard post-electrophoretic staining approaches include Coomassie Brilliant Blue, silver, and a variety of fluorescent dyes. For the efficient pre-electrophoretic comparative labeling of
DIGE-Based Proteomics
19
DIGE-based Proteomics Unbiased large-scale and technology-driven comparative biochemical analysis Crude extracts
Subcellular fractions
Protein fractions
Protein complexes
Comparative 2D-DIGE analysis of differentially labelled protein fractions
Advantages -
-
-
Robust protein separation system for the routine analysis of thousands of protein species in largescale proteomic studies Rapid, cost-effective and highly reproducible approach to study paired protein samples Elimination of gel-to-gel variations by the differential pre-electrophoretic labelling of proteins and the subsequent separation on the same 2D gel Fluorescent labelling with highly sensitive dyes for the visualization of a wide dynamic range of proteins of differing abundance Bioanalytical platform that is suitable for being combined with other dyes and PTM analysis Direct visualization of proteins of interest as discrete 2D spots, enabling the exact evaluation of the characteristic combination of the pI-value and relative molecular mass of a particular protein
Limitations -
-
Under-representation of certain protein species in 2D-DIGE gels: - highly hydrophobic proteins - low-copy-number proteins - high-molecular-mass proteins - proteins with extremely low or high pI-values Restricted separation of complex protein mixtures due to streaking within 2D gel system Cross-contamination of individual 2D protein spots through highly abundant polypeptides Potential complications of MSbased protein identification based on the heterogeneous composition of some 2D protein spots
Fig. 1 Overview of the advantages versus technical limitations of DIGE analysis. Listed are advantages versus technical limitations of the comparative difference gel electrophoresis (DIGE) analysis of preelectrophoretically labeled protein fractions
different protein samples, fluorescence DIGE analysis represents one of the most powerful methods [12]. The DIGE method can be carried out with 2-CyDye or 3-CyDye systems to differentially label proteins belonging to dissimilar protein populations [13]. General advantages versus technical limitations of DIGE analyses are summarized in Fig. 1. The advantages of DIGE over other protein separation methods are its robustness for the routine analysis of thousands of protein species, the cost-effective and highly reproducible nature of fluorescence gel electrophoresis, the elimination of gel-to-gel variations and the direct visualization of a wide dynamic range of proteins of differing abundance, as well as the capability of being combined with other protein staining methods and post-translational modification analysis. Potential limitations are presented by the possible under-representation of certain protein species in DIGE gels, including highly hydrophobic proteins, low-copy-number proteins, high-molecular-mass proteins and protein species with extremely low or high pI-values, the restricted separation of complex protein mixtures, and the crosscontamination of individual protein spots through highly abundant polypeptides [14].
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Proteomic DIGE Workflow A typical DIGE-based proteomic workflow for the comparative analysis of differing proteomes involves several critical steps, including (1) efficient protein extraction from samples derived from defined biological specimens, such as biofluids, tissue extracts, or subcellular fractions, (2) the pre-electrophoretic fluorescent labeling employing 2-dye or 3-dye fluorescence systems with minimal or saturation labeling, (3) effective protein separation by two-dimensional gel electrophoresis using optimized combinations of isoelectric focusing gels and second-dimension slab gels, (4) densitometric scanning of fluorescent images using computer-assisted analysis for the generation of meaningful proteomic maps, (5) optimized protein digestion for the generation of representative peptide signatures, such as the consecutive application of the enzymes Lys-C and trypsin for the controlled and highly reproducible production of peptide populations, and (6) the proteomic identification of individual proteins of interest by sensitive mass spectrometric analysis [15]. Figure 2 outlines the usage of 2-dye versus 3-dye systems in
3-Dye DIGE Sample
2-Dye DIGE
Pooled samples
Sample
Sample
B
A
Pooled samples
Sample
A
Cy3 Dye
Cy2 Dye
Cy5 Dye
Cy3 Dye
Cy5 Dye
Cy3 Dye
Pre-electrophoretic protein labelling
B
Pre-electrophoretic protein labelling
2D IEF/SDS-PAGE Separation
2D IEF/SDS-PAGE Separation
Co-migration of labelled proteins
Co-migration of labelled proteins
Scanning of Cy3-Cy2-Cy5 Images
Scanning of Scanning of Cy3-Cy5 Images Cy3-Cy5 Images
Image analysis of differential spot pattern
Image analysis of differential spot pattern
Identification of altered protein spots
Identification of altered protein spots
Fig. 2 Outline of 2-dye versus 3-dye DIGE analysis. Shown are flowcharts of routine comparative difference gel electrophoresis (DIGE) analyses of pre-electrophoretically labeled protein fractions using fluorescent Cy2, Cy3, and Cy5 dyes
DIGE-Based Proteomics
21
comparative DIGE studies. Often DIGE-generated data are independently verified by routine biochemical, cell biological or biophysical assays, including immunoblotting, enzyme-linked immunosorbent assays, enzyme testing, binding assays, physiological measurements, and/or immunofluorescence microscopy.
4
DIGE Labeling Approaches The comparative DIGE method has been applied in a variety of biological and biomedical research areas, including microbiology, environmental sciences, plant science, animal science, biomedicine, pharmaco-proteomics, and biomarker research, to improve diagnostics, prognostics, therapy-monitoring, and the evaluation of potentially harmful side effects [14]. Routine DIGE analyses are usually carried out with two different approaches, i.e., minimal or saturation protein labeling employing the pre-electrophoretic labeling with fluorescent 2-dye or 3-dye systems [16–18]. The method was originally developed by Unl€ u et al. [19] and is now one of the most frequently used gel-based techniques of comparative proteomics. Sophisticated 2D-DIGE software analysis tools are available to study paired protein samples [20] and are extremely useful for the routine quantitative analysis of multiple fluorescently labeled protein populations following high-resolution and two-dimensional gel electrophoretic separation [21]. Comparative DIGE experiments are routinely carried out with two different dye labeling approaches, i.e., minimal labeling versus saturation labeling. Major differences in the two methods are diagrammatically summarized in Fig. 3. Minimal labeling chemistry is based on N-hydroxy succinimidyl ester dye reagents that form sub-stochiometric bonds with the ε-amine groups of assessable lysine side chains. In contrast to the minimal dye coupling process that involves a nucleophilic substitution reaction, saturation labeling is performed with maleimide dye reagents and the complete labeling of all cysteine sulfhydryls [14]. A 3-sample comparison is shown in Fig. 4, summarizing the labeling approach with the least number of gels to achieve the evaluation of reverse labeling, sample pairing, and biological repeats. If two sets of six DIGE gels are run in parallel, this 12-gel DIGE system can be electrophoresed in the same buffer tank and achieve optimum results in relation to biological repeats (n ¼ 4), sample pairing (n ¼ 2), technical repeats (n ¼ 2), and reverse fluorescent dye labeling (n ¼ 2), as previously described for the comparison of normal, diseased, and therapeutically treated diaphragm muscle specimens [22].
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Kay Ohlendieck
Minimal labelling 2D-DIGE
Saturation labelling 2D-DIGE Pre-electrophoretic differential protein labelling Cy3 Cy5
Pre-electrophoretic differential protein labelling Cy2 Cy3 Cy5
PROTEIN
PROTEIN
Lysine
50 µg protein / 400 pmol dye
pH 8.5 30 min on ice
Excess dye
DYE
Quenching of labeling reaction with excess lysine
DYE
DYE
Sub-stochiometrically labelled proteins 2D-GE
pH 6.5 Saturation labeling
Complete dye labelling of all cysteine residues
Dye labelling of 1-2% of lysine residues
PROTEIN
Cysteine
Separation
DYE
PROTEIN
DYE
Saturation labelled proteins 2D-GE
Separation
Differential 2D spot pattern
Differential 2D spot pattern
Proteomic analysis
Proteomic analysis
Fig. 3 Overview of minimal versus saturation DIGE labeling. Shown are flowcharts that compare the minimal labeling difference gel electrophoresis (DIGE) approach with pre-electrophoretic differential protein labeling using Cy2, Cy3, and Cy5 dyes, and the saturation labeling DIGE approach with pre-electrophoretic differential protein labeling using Cy3 and Cy5 dyes
5
Conclusions The many successful applications of the DIGE technique in combination with advanced mass spectrometry emphasize the analytical power of this comparative proteomic approach. DIGE has been employed in many areas of biological research, biotechnology, biomedicine, and biomarker discovery. The bioanalytical robustness of the DIGE technique makes this biochemical method an ideal comparative tool for the comprehensive analysis of large and complex proteomes. The elimination of gel-to-gel variations and the capability of using DIGE in combination with the analysis of post-translational modifications established this fluorescent gelbased method as a key technique of comparative proteomics. Future applications of comparative DIGE analyses in combination with sensitive protein identification approaches promise to further improve our biochemical understanding of complex proteomes in a variety of biological systems.
DIGE-Based Proteomics
23
3-Sample Comparison using a 6-gel DIGE System Gel 1
Sample A1 Cy3
Standard Cy2
Sample B1 Cy5
Gel 2
Sample C1 Cy3
Standard Cy2
Sample A2 Cy5
Gel 3
Sample B2 Cy3
Standard Cy2
Sample C2 Cy5
Gel 4
Sample A3 Cy3
Standard Cy2
Sample B3 Cy5
Gel 5
Sample C3 Cy3
Standard Cy2
Sample A4 Cy5
Gel 6
Sample B4 Cy3
Standard Cy2
Sample C4 Cy5
Biological Repeats
Technical Repeats
Sample pairing
Reverse labelling
Fig. 4 Outline of 3-sample DIGE analysis. Shown is the combination of 3 different biological samples (A1-A4, B1-B4, and C1-C4) and 3 fluorescent dyes (Cy2, Cy3, and Cy5) for the routine comparative difference gel electrophoresis (DIGE) analyses of pre-electrophoretically labeled protein fractions using a 6-gel system. If two sets of six DIGE gels are run in parallel, a 12-gel DIGE system can achieve optimum results in relation to biological repeats (n ¼ 4), sample pairing (n ¼ 2), technical repeats (n ¼ 2), and reverse fluorescent dye labeling (n ¼ 2)
Acknowledgements Research in the author’s laboratory has been supported by project grants from the Irish Higher Education Authority, the Irish Health Research Board, and Muscular Dystrophy Ireland. References 1. Brewis IA, Brennan P (2010) Proteomics technologies for the global identification and quantification of proteins. Adv Protein Chem Struct Biol 80:1–44 2. Angel TE, Aryal UK, Hengel SM et al (2012) Mass spectrometry-based proteomics: existing capabilities and future directions. Chem Soc Rev 41:3912–3928
3. Zhang Z, Wu S, Stenoien DL et al (2014) High-throughput proteomics. Annu Rev Anal Chem 7:427–454 4. Friedman DB, Hoving S, Westermeier R (2009) Isoelectric focusing and twodimensional gel electrophoresis. Methods Enzymol 463:515–540
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5. Go¨rg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4:3665–3685 6. Carrette O, Burkhard PR, Sanchez JC, Hochstrasser DF (2006) State-of-the-art twodimensional gel electrophoresis: a key tool of proteomics research. Nat Protoc 1:812–823 7. Rabilloud T, Lelong C (2011) Twodimensional gel electrophoresis in proteomics: a tutorial. J Proteomics 74:1829–1841 8. Rabilloud T, Chevallet M, Luche S, Lelong C (2010) Two-dimensional gel electrophoresis in proteomics: past, present and future. J Proteomics 73:2064–2077 9. Oliveira BM, Coorssen JR, Martins-de-Souza D (2014) 2DE: the phoenix of proteomics. J Proteomics 104:140–150 10. Murphy S, Dowling P, Ohlendieck K (2016) Comparative skeletal muscle proteomics using two-dimensional gel electrophoresis. Proteomes 4:27 11. Timms JF, Cramer R (2008) Difference gel electrophoresis. Proteomics 8:4886–4897 12. Viswanathan S, Unl€ u M, Minden JS (2006) Two-dimensional difference gel electrophoresis. Nat Protoc 1:1351–1358 13. Alban A, David SO, Bjorkesten L et al (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3:36–44 14. Arentz G, Weiland F, Oehler MK et al (2015) State of the art of 2D DIGE. Proteomics Clin Appl 9:277–288
15. Minden JS, Dowd SR, Meyer HE et al (2009) Difference gel electrophoresis. Electrophoresis 30:S156–S161 16. Karp NA, Kreil DP, Lilley KS (2004) Determining a significant change in protein expression with DeCyder during a pair-wise comparison using two-dimensional difference gel electrophoresis. Proteomics 4:1421–1432 17. Karp NA, Lilley KS (2005) Maximising sensitivity for detecting changes in protein expression: experimental design using minimal CyDyes. Proteomics 5:3105–3115 18. Carberry S, Zweyer M, Swandulla D et al (2013) Application of fluorescence twodimensional difference in-gel electrophoresis as a proteomic biomarker discovery tool in muscular dystrophy research. Biology (Basel) 2:1438–1464 19. Unl€ u M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18:2071–2077 20. Tonge R, Shaw J, Middleton B et al (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377–396 21. Marouga R, David S, Hawkins E (2005) The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal Bioanal Chem 382:669–678 22. Doran P, Wilton SD, Fletcher S et al (2009) Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm. Proteomics 9:671–685
1
Introduction The successful isolation, identification, and detailed characterization of individual protein species, complex protein assemblies, organellar subproteomes, or entire proteomes by analytical biochemistry rely heavily on the appropriate combination of a variety of standardized techniques. The routine workflow in many protein biochemical and proteomic studies includes: (1) the efficient extraction of proteins with widely differing physicochemical properties from complex biological samples, (2) the careful protection of sensitive proteins from degradation during lengthy isolation procedures, (3) the basic separation of structurally or functionally related protein populations by relatively crude subcellular fractionation methods such as differential or density gradient centrifugation, (4) the refined separation of different protein classes by the systematic application of large-scale methods such as gel
Kay Ohlendieck (ed.), Difference Gel Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 1664, DOI 10.1007/978-1-4939-7268-5_2, © Springer Science+Business Media LLC 2018
17
18
Kay Ohlendieck
electrophoresis, liquid chromatography, and/or affinity methods, (5) the reliable identification of representative peptides, individual protein fragments or intact proteins by mass spectrometry or antibody-based techniques, (6) the swift determination of protein concentration and isoform expression patterns, and (7) the comprehensive determination of post-translational modifications [1–3]. Over the last few decades of bioresearch, gel electrophoresis has played a central role in both preparative and analytical terms [4] and has been widely employed for efficient peptide and protein separation in the more recently established field of mass spectrometry-based proteomics [5–7]. Although a variety of liquid chromatographic methods are now frequently incorporated in proteomic studies, gel electrophoretic techniques are at the core of modern proteomics and routinely employed in the systems biological analysis of proteome-wide changes and adaptations in health and disease [8–10]. Many different electrophoretic approaches are employed in analytical biochemistry, whereby difference gel electrophoresis (DIGE) presents one of the most powerful comparative techniques in modern proteomics [11]. Using particular DIGE-based methods, the efficient gel electrophoretic separation of proteins is mostly based on natural or modified differences in overall charge between individual polypeptide chains, as well as dissimilarities in molecular size under native or denatured conditions. This chapter briefly outlines the development, technical advances, and applications of DIGE in modern proteomics.
2
Gel-Based Proteomics and DIGE Analysis In general, gel electrophoretic techniques can be distinguished based on the dimensionality of the gel system and the main labeling procedure for the visualization of protein bands or spot patterns. Routinely used one-dimensional gel electrophoretic methods for the separation of proteins include isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis [4]. A combination of both techniques in two-dimensional gel electrophoresis is significantly more efficient for large-scale protein separation approaches due to its huge capacity and high resolution, making it a frequently used method in high-throughput proteomic surveys [8]. Depending on the particular gel electrophoretic technique and the analyzed samples, several hundred to thousands of individual protein spots can be visualized by standardized two-dimensional gel electrophoresis [5]. Besides optimizing the electrophoretic procedure, protein labeling is a crucial step in gel-based analytical proteomics. Standard post-electrophoretic staining approaches include Coomassie Brilliant Blue, silver, and a variety of fluorescent dyes. For the efficient pre-electrophoretic comparative labeling of
DIGE-Based Proteomics
19
DIGE-based Proteomics Unbiased large-scale and technology-driven comparative biochemical analysis Crude extracts
Subcellular fractions
Protein fractions
Protein complexes
Comparative 2D-DIGE analysis of differentially labelled protein fractions
Advantages -
-
-
Robust protein separation system for the routine analysis of thousands of protein species in largescale proteomic studies Rapid, cost-effective and highly reproducible approach to study paired protein samples Elimination of gel-to-gel variations by the differential pre-electrophoretic labelling of proteins and the subsequent separation on the same 2D gel Fluorescent labelling with highly sensitive dyes for the visualization of a wide dynamic range of proteins of differing abundance Bioanalytical platform that is suitable for being combined with other dyes and PTM analysis Direct visualization of proteins of interest as discrete 2D spots, enabling the exact evaluation of the characteristic combination of the pI-value and relative molecular mass of a particular protein
Limitations -
-
Under-representation of certain protein species in 2D-DIGE gels: - highly hydrophobic proteins - low-copy-number proteins - high-molecular-mass proteins - proteins with extremely low or high pI-values Restricted separation of complex protein mixtures due to streaking within 2D gel system Cross-contamination of individual 2D protein spots through highly abundant polypeptides Potential complications of MSbased protein identification based on the heterogeneous composition of some 2D protein spots
Fig. 1 Overview of the advantages versus technical limitations of DIGE analysis. Listed are advantages versus technical limitations of the comparative difference gel electrophoresis (DIGE) analysis of preelectrophoretically labeled protein fractions
different protein samples, fluorescence DIGE analysis represents one of the most powerful methods [12]. The DIGE method can be carried out with 2-CyDye or 3-CyDye systems to differentially label proteins belonging to dissimilar protein populations [13]. General advantages versus technical limitations of DIGE analyses are summarized in Fig. 1. The advantages of DIGE over other protein separation methods are its robustness for the routine analysis of thousands of protein species, the cost-effective and highly reproducible nature of fluorescence gel electrophoresis, the elimination of gel-to-gel variations and the direct visualization of a wide dynamic range of proteins of differing abundance, as well as the capability of being combined with other protein staining methods and post-translational modification analysis. Potential limitations are presented by the possible under-representation of certain protein species in DIGE gels, including highly hydrophobic proteins, low-copy-number proteins, high-molecular-mass proteins and protein species with extremely low or high pI-values, the restricted separation of complex protein mixtures, and the crosscontamination of individual protein spots through highly abundant polypeptides [14].
20
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Kay Ohlendieck
Proteomic DIGE Workflow A typical DIGE-based proteomic workflow for the comparative analysis of differing proteomes involves several critical steps, including (1) efficient protein extraction from samples derived from defined biological specimens, such as biofluids, tissue extracts, or subcellular fractions, (2) the pre-electrophoretic fluorescent labeling employing 2-dye or 3-dye fluorescence systems with minimal or saturation labeling, (3) effective protein separation by two-dimensional gel electrophoresis using optimized combinations of isoelectric focusing gels and second-dimension slab gels, (4) densitometric scanning of fluorescent images using computer-assisted analysis for the generation of meaningful proteomic maps, (5) optimized protein digestion for the generation of representative peptide signatures, such as the consecutive application of the enzymes Lys-C and trypsin for the controlled and highly reproducible production of peptide populations, and (6) the proteomic identification of individual proteins of interest by sensitive mass spectrometric analysis [15]. Figure 2 outlines the usage of 2-dye versus 3-dye systems in
3-Dye DIGE Sample
2-Dye DIGE
Pooled samples
Sample
Sample
B
A
Pooled samples
Sample
A
Cy3 Dye
Cy2 Dye
Cy5 Dye
Cy3 Dye
Cy5 Dye
Cy3 Dye
Pre-electrophoretic protein labelling
B
Pre-electrophoretic protein labelling
2D IEF/SDS-PAGE Separation
2D IEF/SDS-PAGE Separation
Co-migration of labelled proteins
Co-migration of labelled proteins
Scanning of Cy3-Cy2-Cy5 Images
Scanning of Scanning of Cy3-Cy5 Images Cy3-Cy5 Images
Image analysis of differential spot pattern
Image analysis of differential spot pattern
Identification of altered protein spots
Identification of altered protein spots
Fig. 2 Outline of 2-dye versus 3-dye DIGE analysis. Shown are flowcharts of routine comparative difference gel electrophoresis (DIGE) analyses of pre-electrophoretically labeled protein fractions using fluorescent Cy2, Cy3, and Cy5 dyes
DIGE-Based Proteomics
21
comparative DIGE studies. Often DIGE-generated data are independently verified by routine biochemical, cell biological or biophysical assays, including immunoblotting, enzyme-linked immunosorbent assays, enzyme testing, binding assays, physiological measurements, and/or immunofluorescence microscopy.
4
DIGE Labeling Approaches The comparative DIGE method has been applied in a variety of biological and biomedical research areas, including microbiology, environmental sciences, plant science, animal science, biomedicine, pharmaco-proteomics, and biomarker research, to improve diagnostics, prognostics, therapy-monitoring, and the evaluation of potentially harmful side effects [14]. Routine DIGE analyses are usually carried out with two different approaches, i.e., minimal or saturation protein labeling employing the pre-electrophoretic labeling with fluorescent 2-dye or 3-dye systems [16–18]. The method was originally developed by Unl€ u et al. [19] and is now one of the most frequently used gel-based techniques of comparative proteomics. Sophisticated 2D-DIGE software analysis tools are available to study paired protein samples [20] and are extremely useful for the routine quantitative analysis of multiple fluorescently labeled protein populations following high-resolution and two-dimensional gel electrophoretic separation [21]. Comparative DIGE experiments are routinely carried out with two different dye labeling approaches, i.e., minimal labeling versus saturation labeling. Major differences in the two methods are diagrammatically summarized in Fig. 3. Minimal labeling chemistry is based on N-hydroxy succinimidyl ester dye reagents that form sub-stochiometric bonds with the ε-amine groups of assessable lysine side chains. In contrast to the minimal dye coupling process that involves a nucleophilic substitution reaction, saturation labeling is performed with maleimide dye reagents and the complete labeling of all cysteine sulfhydryls [14]. A 3-sample comparison is shown in Fig. 4, summarizing the labeling approach with the least number of gels to achieve the evaluation of reverse labeling, sample pairing, and biological repeats. If two sets of six DIGE gels are run in parallel, this 12-gel DIGE system can be electrophoresed in the same buffer tank and achieve optimum results in relation to biological repeats (n ¼ 4), sample pairing (n ¼ 2), technical repeats (n ¼ 2), and reverse fluorescent dye labeling (n ¼ 2), as previously described for the comparison of normal, diseased, and therapeutically treated diaphragm muscle specimens [22].
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Kay Ohlendieck
Minimal labelling 2D-DIGE
Saturation labelling 2D-DIGE Pre-electrophoretic differential protein labelling Cy3 Cy5
Pre-electrophoretic differential protein labelling Cy2 Cy3 Cy5
PROTEIN
PROTEIN
Lysine
50 µg protein / 400 pmol dye
pH 8.5 30 min on ice
Excess dye
DYE
Quenching of labeling reaction with excess lysine
DYE
DYE
Sub-stochiometrically labelled proteins 2D-GE
pH 6.5 Saturation labeling
Complete dye labelling of all cysteine residues
Dye labelling of 1-2% of lysine residues
PROTEIN
Cysteine
Separation
DYE
PROTEIN
DYE
Saturation labelled proteins 2D-GE
Separation
Differential 2D spot pattern
Differential 2D spot pattern
Proteomic analysis
Proteomic analysis
Fig. 3 Overview of minimal versus saturation DIGE labeling. Shown are flowcharts that compare the minimal labeling difference gel electrophoresis (DIGE) approach with pre-electrophoretic differential protein labeling using Cy2, Cy3, and Cy5 dyes, and the saturation labeling DIGE approach with pre-electrophoretic differential protein labeling using Cy3 and Cy5 dyes
5
Conclusions The many successful applications of the DIGE technique in combination with advanced mass spectrometry emphasize the analytical power of this comparative proteomic approach. DIGE has been employed in many areas of biological research, biotechnology, biomedicine, and biomarker discovery. The bioanalytical robustness of the DIGE technique makes this biochemical method an ideal comparative tool for the comprehensive analysis of large and complex proteomes. The elimination of gel-to-gel variations and the capability of using DIGE in combination with the analysis of post-translational modifications established this fluorescent gelbased method as a key technique of comparative proteomics. Future applications of comparative DIGE analyses in combination with sensitive protein identification approaches promise to further improve our biochemical understanding of complex proteomes in a variety of biological systems.
DIGE-Based Proteomics
23
3-Sample Comparison using a 6-gel DIGE System Gel 1
Sample A1 Cy3
Standard Cy2
Sample B1 Cy5
Gel 2
Sample C1 Cy3
Standard Cy2
Sample A2 Cy5
Gel 3
Sample B2 Cy3
Standard Cy2
Sample C2 Cy5
Gel 4
Sample A3 Cy3
Standard Cy2
Sample B3 Cy5
Gel 5
Sample C3 Cy3
Standard Cy2
Sample A4 Cy5
Gel 6
Sample B4 Cy3
Standard Cy2
Sample C4 Cy5
Biological Repeats
Technical Repeats
Sample pairing
Reverse labelling
Fig. 4 Outline of 3-sample DIGE analysis. Shown is the combination of 3 different biological samples (A1-A4, B1-B4, and C1-C4) and 3 fluorescent dyes (Cy2, Cy3, and Cy5) for the routine comparative difference gel electrophoresis (DIGE) analyses of pre-electrophoretically labeled protein fractions using a 6-gel system. If two sets of six DIGE gels are run in parallel, a 12-gel DIGE system can achieve optimum results in relation to biological repeats (n ¼ 4), sample pairing (n ¼ 2), technical repeats (n ¼ 2), and reverse fluorescent dye labeling (n ¼ 2)
Acknowledgements Research in the author’s laboratory has been supported by project grants from the Irish Higher Education Authority, the Irish Health Research Board, and Muscular Dystrophy Ireland. References 1. Brewis IA, Brennan P (2010) Proteomics technologies for the global identification and quantification of proteins. Adv Protein Chem Struct Biol 80:1–44 2. Angel TE, Aryal UK, Hengel SM et al (2012) Mass spectrometry-based proteomics: existing capabilities and future directions. Chem Soc Rev 41:3912–3928
3. Zhang Z, Wu S, Stenoien DL et al (2014) High-throughput proteomics. Annu Rev Anal Chem 7:427–454 4. Friedman DB, Hoving S, Westermeier R (2009) Isoelectric focusing and twodimensional gel electrophoresis. Methods Enzymol 463:515–540
24
Kay Ohlendieck
5. Go¨rg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4:3665–3685 6. Carrette O, Burkhard PR, Sanchez JC, Hochstrasser DF (2006) State-of-the-art twodimensional gel electrophoresis: a key tool of proteomics research. Nat Protoc 1:812–823 7. Rabilloud T, Lelong C (2011) Twodimensional gel electrophoresis in proteomics: a tutorial. J Proteomics 74:1829–1841 8. Rabilloud T, Chevallet M, Luche S, Lelong C (2010) Two-dimensional gel electrophoresis in proteomics: past, present and future. J Proteomics 73:2064–2077 9. Oliveira BM, Coorssen JR, Martins-de-Souza D (2014) 2DE: the phoenix of proteomics. J Proteomics 104:140–150 10. Murphy S, Dowling P, Ohlendieck K (2016) Comparative skeletal muscle proteomics using two-dimensional gel electrophoresis. Proteomes 4:27 11. Timms JF, Cramer R (2008) Difference gel electrophoresis. Proteomics 8:4886–4897 12. Viswanathan S, Unl€ u M, Minden JS (2006) Two-dimensional difference gel electrophoresis. Nat Protoc 1:1351–1358 13. Alban A, David SO, Bjorkesten L et al (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3:36–44 14. Arentz G, Weiland F, Oehler MK et al (2015) State of the art of 2D DIGE. Proteomics Clin Appl 9:277–288
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