The evolution of two-dimensional gel electrophoresis - from proteomics to emerging alternative applications.

Two-dimensional gel electrophoresis (2-DE) is a technique that has been widely applied in a variety of proteomics studies. It is capable of resolving complex protein mixtures into individual protein spots based on their isoelectric point and molecular weight, enabling large-scale analysis of protein expression patterns for deciphering their changes in different biological conditions. 2-DE is a powerful tool that empowers researchers to perform differential qualitative and quantitative proteome analysis and is particularly advantageous for characterizing protein isoforms and post-translationally modified proteins. Despite its popularity as the workhorse for proteomics in the past few decades, it has been gradually displaced by the more sophisticated and high-performance mass spectrometry-based methods. However, there are several variations of the 2-DE technique that have emerged as promising approaches that shine new light on specific niches that 2-DE could still contribute. In this review, we first provide an overview of the applications of 2-DE, its merits and pitfalls in the current proteomic research arena, followed by a discussion on several alternative approaches for potential future applications.

Cancer biomarker development and two-dimensional difference gel electrophoresis (2D-DIGE).

Cancer results from the accumulation of genomic alterations. As the genome is functionally translated to the proteome and regulates tumor cell behavior, proteomics studies are expected to further the current understanding of the molecular mechanisms underlying carcinogenesis and cancer progression. Biomarkers are potential tools to classify cancers for therapy, predict responses to treatments, and support treatment-related decision-making. Biomarker development has been actively pursued in oncology by proteomic approaches. Two-dimensional difference gel electrophoresis (2D-DIGE) is a proteomics technique based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In 2D-DIGE, protein samples are labeled with distinct fluorescent dyes before fractionation via 2D-PAGE. 2D-DIGE offers advantages to identify biomarker candidates, including reproducibility, high sensitivity, comprehensiveness, and high throughput. 2D-DIGE has contributed to the establishment of tissue biomarkers, which potentially facilitate precision medicine. 2D-DIGE is thus expected to yield major advancements in cancer biomarker identification and development.

Gel-based proteomics in disease research: Is it still valuable?

Gel electrophoresis had been the primary method in proteomics. In the early era of proteomics, gel electrophoresis was a dominant technique of sample preparation for mass spectrometry analysis. Particularly, two-dimensional electrophoresis provided high-resolution proteome separation, and was regarded as the standard methodology for the separation of wide-range proteomes. However, gel electrophoresis turned downwards due to the progress of other separations including liquid chromatography and ionization techniques, resulting gel-free proteomics finally becoming dominant players at present. There are numerous advantages in gel-free approach in aspects of current trends of disease research. Interestingly, gel-free approaches are still advanced, it seems that gel electrophoresis will not be disappeared. The unique features of gel electrophoresis can be complementary for gel-free and it is suitable for the new wave of top-down functional proteomics.

Gel electrophoresis-based plant proteomics: Past, present, and future. Happy 10th anniversary Journal of Proteomics!

In this century we have assisted at an unimaginable expansion of proteomics, with continuous innovations and optimizations in methods, techniques, protocols, equipment, and associated bioinformatics tools. We have moved forward very fast from first (gel electrophoresis based), to second (based on isotopic or isobaric labelling), to third (shotgun or gel-free, label-free), and to fourth (targeted, mass-western, or SRM/MRM) generation techniques. This evolution is clearly observed in the literature since 1994, when the term "proteome" was first coined, with plant proteomics progressing at a much lower speed than human and other model organisms. The question behind this review is: Is gel electrophoresis an obsolete technique? Is it still alive? The answer is that gel electrophoresis is still a valid technique, with its own particularities, strengths, and weaknesses, "irreplaceable" in top-down experiments directed at investigating protein species, loci and allelic variants, and isoforms, as well as in the post-translational modifications and interactions studies; it is an excellent complementary and alternative approach that could lead us to achieve a deeper visualization and knowledge of the cell proteome. The past, present, and future of this technique is being reviewed. It is not pretended to discuss in detail technical aspects, referring to key original papers or previous reviews, but instead, how it has contributed, from a historical perspective, to plant proteomics and biology research. It is our personal congratulations to "Journal of Proteomics" that celebrates this year its 10th anniversary, and, at the same time, a tribute to those scientists who have contributed to the establishment and development of the gel electrophoresis technique and its application to proteomics and plant biology research. Their direct or indirect teaching has been very valuable to those of us who once decided to enter proteomics, with no access to any sophisticated and expensive equipment. This gel electrophoresis-based plant proteomics review is divided into the following sections: introduction, history, methodology, contribution to plant biology research, and future directions.

Identification of plantago lanceolata pollen allergens using an immunoproteomic approach

Background: Airborne Plantago pollen triggers respiratory allergies in Mediterranean countries. Objectives: We aimed to study sensitization in patients with seasonal respiratory allergy and identify proteins of Plantago lanceolata pollen that could be responsible for hypersensitivity reactions in sensitized patients. We also determined the airborne pollen concentration of Plantago species from 2004 to 2011. Methods: IgE-binding proteins were analyzed and characterized using 1D and 2D gel electrophoresis and immunoblotting with sera from individuals sensitized to P lanceolata pollen extracts, mass spectrometry analysis, and protein data mining. We used aerobiological methods to study airborne pollen. Results: P lanceolata pollen accounts for 3% of the annual pollen spectrum in the air of Porto. Of a total of 372 patients, 115 (31%) showed specific IgE levels to P lanceolata pollen extracts. All sera from P lanceolata –allergic patients recognized 8 prominent groups of IgE-reactive allergens. Separation of proteins using 2D gel electrophoresis followed by identification with mass spectrometry revealed the presence of other IgE-reactive components that could be involved in sensitization. Conclusions: We detected proteins in P lanceolata pollen extracts that, to our knowledge, have not yet been studied and could worsen sensitization to this weed pollen species. The proteins identified were involved in a variety of cellular functions. By applying 2D electrophoresis and immunoblotting with a pool of 2 sera from different P lanceolata -allergic patients, we obtained a more detailed characterization of the P lanceolata allergen profile (AU)

Antecedentes: El polen de Plantago provoca alergia respiratoria en los países mediterráneos. Objetivos: El objetivo de este estudio fue analizar las sensibilizaciones de pacientes con alergia estacional e identificar las proteínas de polen de Plantago lanceolata que puedan ser responsables de las reacciones de hipersensibilidad en pacientes sensibles. Adicionalmente determinamos la concentración de polen de Plantago spp en el aire, en los años 2004-2011. Métodos: Las proteínas que se unen a la IgE fueron analizadas y caracterizadas a través de electroforesis en gel 1-D y 2-D e inmunobloting con suero de pacientes sensibilizados al polen de P. lanceolata . Se analizó mediante espectrometría de masas el contenido en las proteínas y se aplicaron métodos aerobiológicos para estudiar el espectro de polen en el ambiente. Resultados: En cuanto a los resultados obtenidos, el polen de P. lanceolata representa el 3% del espectro de polen anual en la atmósfera de Oporto. De los 372 pacientes, el 31% presentaban IgE específica frente al polen de P. lanceolata. Todos los sueros de los pacientes alérgicos a P. Lanceolata reconocían los ocho grupos prominentes de alérgenos reactivos a IgE. La separación de proteínas mediante electroforesis en gel 2-D, seguida de la espectrofotometría de masas permitieron identificar en el polen la presencia de otros componentes IgE reactivos que podrían estar implicados en la sensibilización de estos pacientes. Conclusiones: En conclusión, este estudio muestra la presencia de proteínas en el polen de P. Lanceolata que hasta ahora no habían sido estudiadas y que pueden intervenir en la sensibilización a éste polen. Se detectaron proteínas involucradas en una gran variedad de funciones celulares. Mediante las técnicas aplicadas en este estudio, entre ellas el inmunobloting, nos permite realizar una detallada caracterización del perfil alergénico del polen de P. lanceolata (AU)

Fosfoglicerato quinasa y fructosa bifosfato aldolasa de Candida albicans como nuevos antígenos reconocidos por la IgA salival humana / Phosphoglycerate kinase and fructose bisphosphate aldolase of Candida albicans as new antigens recognized by human salivary IgA

Fundamento. Candida albicans es un hongo dimórfico oportunista que, con frecuencia, está presente en la cavidad oral del ser humano donde da lugar a infecciones en pacientes inmunocomprometidos. Influida por las condiciones de crecimiento, la variabilidad antigénica es un factor de patogenicidad. Objetivos. Determinar el efecto del estrés nutricional y térmica en la expresión antigénica de C. albicans, e identificar los principales antígenos reconocidos por la inmunoglobulina secretora A (sIgA) salival humana. Métodos. En diferentes condiciones nutricionales, se indujo un choque térmico en células de C. albicans en fase de crecimiento estacionario y exponencial. La expresión de los determinantes proteicos de C. albicans se analizó mediante inmunotransferencia frente a la saliva humana. Los antígenos se purificaron y caracterizaron mediante electroforesis bidimensional y se identificaron mediante microsecuenciación de proteínas. Resultados. Se caracterizaron cinco antígenos reconocidos por la IgA salival como manoproteínas debido a su reactividad con la concanavalina A. Ninguno manifestó reactividad con los anticuerpos monoclonales anti-proteína de choque térmico. Se encontró que el choque térmico y el estrés nutricional regulaban dos de ellos (de 42 y 36 kDa) identificados como fosfoglicerato quinasa y fructosa bifosfato aldolasa, respectivamente. Conclusión. Estas enzimas glucolíticas son antígenos mayores de C. albicans, y su expresión diferencial y el reconocimiento por el sistema de la respuesta inmunitaria de la mucosa podrían participar en la protección frente a las infecciones orales(AU)

Background. Candida albicans is an opportunistic dimorphic fungus commonly present in the human oral cavity that causes infections in immunocompromised patients. The antigen variability, influenced by growth conditions, is a pathogenicity factor. Aims. To determine the effect of nutritional and heat stress on the antigen expression of C. albicans, and to identify major antigens recognized by human salivary secretory immunoglobulin A (sIgA). Methods. Under various different nutritional conditions, heat shock was induced in C. albicans cells in stationary and exponential growth phases. The expression of protein determinants of C. albicans was assessed by Western blot analysis against human saliva. The antigens were purified and characterized by two-dimensional electrophoresis and identified by protein microsequencing. Results. Five antigens recognized by salivary IgA were characterized as mannoproteins due to their reactivity with concanavalin A. They did not show reactivity with anti-heat shock protein monoclonal antibodies. Two of them (42 and 36kDa) were found to be regulated by heat shock and by nutritional stress and they were identified as phosphoglycerate kinase and fructose bisphosphate aldolase, respectively. Conclusions. These glycolytic enzymes are major antigens of C. albicans, and their differential expression and recognition by the mucosal immune response system could be involved in protection against oral infection(AU)

Two-dimensional gel electrophoresis in bacterial proteomics.

Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.

[The application progresses of the two dimensional electrophoresis in biomedical research].

Research about proteomics is of great significance. Two-dimensional electrophoresis (2-DE) is a core technology of proteomics research, which is used for analysis of the protein extracted from cell, tissue and other sam-nology of proteomics research, which is used for analysis of the protein extracted from cell, tissue and other samples. In recent years, 2-DE combined with mass spectrum (MS) technology is widely used to identify differentialples. In recent years, 2-DE combined with mass spectrum (MS) technology is widely used to identify differential protein, to screen tumor markers, to detect drug targets and so on. Proteomics research has become key technology,protein, to screen tumor markers, to detect drug targets and so on. Proteomics research has become key technology, with its high throughput, high resolution and repeatability, and is widely used in various fields, particularly in bio-with its high throughput, high resolution and repeatability, and is widely used in various fields, particularly in biomedical research. We provided here a short review about the application development of 2-DE, especially its contribution on biological medicine.

Two-dimensional gel electrophoresis in proteomics: Past, present and future.

Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned.

Native electrophoretic techniques to identify protein-protein interactions.

Permanent protein-protein interactions are commonly identified by co-purification of two or more protein components using techniques like co-immunoprecipitation, tandem affinity purification and native electrophoresis. Here we focus on blue-native electrophoresis, clear-native electrophoresis, high-resolution clear-native electrophoresis and associated techniques to identify stable membrane protein complexes and detergent-labile physiological supercomplexes. Hints for dynamic protein-protein interactions can be obtained using two-hybrid techniques but not from native electrophoresis and other protein isolation techniques except after covalent cross-linking of interacting proteins in vivo prior to protein separation.

Membrane proteins and proteomics: love is possible, but so difficult.

Despite decades of extensive research, the large-scale analysis of membrane proteins remains a difficult task. This is due to the fact that membrane proteins require a carefully balanced hydrophilic and lipophilic environment, which optimum varies with different proteins, while most protein chemistry methods work mainly, if not only, in water-based media. Taking this review [Santoni, Molloy and Rabilloud, Membrane proteins and proteomics: un amour impossible? Electrophoresis 2000, 21, 1054-1070] as a pivotal paper, the current paper analyzes how the field of membrane proteomics exacerbated the trend in proteomics, i.e. developing alternate methods to the historical two-dimensional electrophoresis, and thus putting more and more pressure on the mass spectrometry side. However, in the case of membrane proteins, the incentive in doing so is due to the poor solubility of membrane proteins. This review also shows that in some situations, where this solubility problem is less acute, two-dimensional electrophoresis remains a method of choice. Last but not least, this review also critically examines the alternate approaches that have been used for the proteomic analysis of membrane proteins.

Challenges, current status and future perspectives of proteomics in improving understanding, diagnosis and treatment of vascular disease.

Technical advances have seen the rapid adoption of genomics and multiplex genetic polymorphism identification to research on vascular diseases. The utilization of proteomics for the study of vascular diseases has been limited by comparison. In this review we outline currently available proteomics techniques, the challenges to using these approaches and modifications which may improve the utilization of proteomics in the study of vascular diseases.

Hares and tortoises: the high- versus low-throughput proteomic race.

The analysis of the proteome can be undertaken with parallel, high-throughput techniques or those that analyze proteins in a serial, one-at-a-time manner. The former include 2-D gels and shotgun MS/MS; the latter includes libraries containing fusion proteins (GST, green fluorescent protein, TAP-tag and others) that are engineered onto each protein in a proteome and then studied one by one. In this review, we explore the progress that these scientifically contrasting paradigms have made in measuring protein abundance, half-life, post-translational modifications, localization in cells and tissues and in protein membership of complexes, pathways and networks. We find that our understanding of the yeast proteome has been furthered more substantially by the slower "tortoise techniques" than the "high-throughput hares". A number of aspects of the human proteome are also likely to be elucidated most accurately with low-throughput approaches. However, the high-throughput techniques are expected to remain crucial for comparative analyses and most studies of proteome dynamics. This review also briefly explores how electrophoretic separations can continue to support the field of proteomics.

Features and applications of blue-native and clear-native electrophoresis.

1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.

Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update.

The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE): advances and perspectives.

The recent trend in science is to assay as many biological molecules as possible within a single experiment. This trend is evident in proteomics where the aim is to characterize thousands of proteins within cells, tissues, and organisms. While advances in mass spectrometry have been critical, developments made in two-dimensional PAGE (2D-PAGE) have also played a major role in enabling proteomics. In this review, we discuss and highlight the advances made in 2D-PAGE over the past 25 years that have made it a foundational tool in proteomic research.

Advanced technologies for studies on protein interactomes.

One of the key challenges of biology in the post-genomic era is to assign function to the many genes revealed by large-scale sequencing programmes, since only a small fraction of gene function can be directly inferred from the coding sequence. Identifying interactions between proteins is a substantial part in understanding their function. The main technologies for investigating protein-protein interactions and assigning functions to proteins include direct detection intermolecular interactions through protein microarray, yeast two-hybrid system, mass spectrometry fluorescent techniques to visualize protein complexes or pull-down assays, as well as technologies detecting functional interactions between genes, such as RNAi knock down or functional screening of cDNA libraries. Over recent years, considerable advances have been made in the above techniques. In this review, we discuss some recent developments and their impact on the gene function annotation.

Comparative proteomics and difference gel electrophoresis.

The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference gel electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE.

Two-dimensional gels for toxicological drug discovery applications.

Two-dimensional gel electrophoresis (2DGE) continues to be a useful approach to study protein expression. Although liquid chromatographic and mass spectrometric approaches that overcome some of the limitations and labour intensity of 2DGE are increasingly popular, this electrophoretic approach still has exceptional relevance in toxicology. Despite the technical challenges, pharmacologists/toxicologists continue to use gel-based proteomics to assess the biological and health effects of chemical treatment and exposure. This brief review addresses the use of 2DGE-based proteomics in drug development and toxicology, emphasising its unique strengths and weaknesses, and considers recent developments in this strategy that have evolved to directly confront the issues of dynamic range and reproducibility that have previously limited the overall use of 2D electrophoresis.

Functional Genomics meets neurodegenerative disorders Part I: transcriptomic and proteomic technology.

Transcriptomics and proteomics are increasingly applied to gain a mechanistic insight into neurodegenerative disorders. These techniques not only identify distinct, differentially expressed mRNAs and proteins but are also employed to dissect signaling pathways and reveal networks by using an integrated approach. In part I of this back-to-back review, technical aspects are discussed: in the transcriptomics section, which includes enrichment by laser microcapture dissection, we comment on qRT-PCR, SAGE, subtractive hybridization, differential display and microarrays, including software packages. In the proteomics section we discuss two-dimensional (2D) gel electrophoresis, liquid chromatography, methods to label and enrich specific proteins or peptides, and different types of mass spectrometers. These tools have been applied to a range of neurodegenerative disorders and are discussed and integrated in part II (Functional Genomics meets neurodegenerative disorders. Part II: application and data integration).