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.

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.

Paleoproteomics explained to youngsters: how did the wedding of two-dimensional electrophoresis and protein sequencing spark proteomics on: let there be light.

Taking the opportunity of the 20th anniversary of the word "proteomics", this young adult age is a good time to remember how proteomics came from enormous progress in protein separation and protein microanalysis techniques, and from the conjugation of these advances into a high performance and streamlined working setup. However, in the history of the almost three decades that encompass the first attempts to perform large scale analysis of proteins to the current high throughput proteomics that we can enjoy now, it is also interesting to underline and to recall how difficult the first decade was. Indeed when the word was cast, the battle was already won. This recollection is mostly devoted to the almost forgotten period where proteomics was being conceived and put to birth, as this collective scientific work will never appear when searched through the keyword "proteomics". BIOLOGICAL SIGNIFICANCE: The significance of this manuscript is to recall and review the two decades that separated the first attempts of performing large scale analysis of proteins from the solid technical corpus that existed when the word "proteomics" was coined twenty years ago. This recollection is made within the scientific historical context of this decade, which also saw the blossoming of DNA cloning and sequencing. This article is part of a Special Issue entitled: 20 years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini , Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

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.

How it all began: a personal history of gel electrophoresis.

Arne Tiselius' moving boundary electrophoresis method was still in general use in 1951 when this personal history begins, although zonal electrophoresis with a variety of supporting media (e.g., filter paper or starch grains) was beginning to replace it. This chapter is an account of 10 years of experiments carried out by the author during which molecular sieving gel electrophoresis was developed and common genetic variants of two proteins, haptoglobin and transferrin, were discovered in normal individuals. Most of the figures are images of pages from the author's laboratory notebooks, which are still available, so that some of the excitement of the time and the humorous moments are perhaps apparent. Alkaline gels, acidic gels with and without denaturants, vertical gels, two-dimensional gels, and gels with differences in starch concentration are presented. The subtle details that can be discerned in these various gels played an indispensable role in determining the nature of the change in the haptoglobin gene (Hp) that leads to the polymeric series characteristic of Hp ( 2 ) /Hp ( 2 ) homozygotes. Where possible, the names of scientific friends who made this saga of gel electrophoresis so memorable and enjoyable are gratefully included.

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.

From 2-D electrophoresis to proteomics.

At first, a short history of the beginning of 2-DE is provided. Based on the present state of the art at the time I developed a 2-DE technique in 1975 that was able to resolve complex protein extracts from mouse tissues in hundreds of protein spots. My intention was to study proteins from a global point of view. Questions of interest were, how do proteins change during embryonic development, and what is the effect of induced mutations on the protein level. At that time protein chemistry was a matter of analyzing single proteins in detail. Therefore, my approach was frequently criticized as inappropriate because it would be impossible to identify and characterize the hundreds of proteins resolved. But soon it was realized that studying total proteins gives opportunities to answer many interesting questions. This led to a research field nowadays called "proteomics". Already in the beginning of the 1980s the idea to analyze the total human proteins had come up. By entering the post-genome era it became obvious that a human proteome project is needed in order to explain the human genome in terms of its functions. The problems in realizing such a project are considered.

Immobilized pH gradients.

In this short review, we give an account of the steps through which the protocols for operation with IPGs were set up in the early '80s. One of the main achievements by our group was the development of a computer program, pH GRADIENT, for the calculation of pH, buffering power and ionic strength of a mixture of monoprotic buffers titrated within any pH span and for the linearization of the desired pH gradient. Using this program, in 1984 we could devise formulations for IPGs covering up to six pH units, which was the subject of a publication in Electrophoresis (volume 5, pages 88-97). This was the starting point for the use of IPGs for the resolution of protein samples of any composition and for their application as first dimension of 2-DE separations. Currently IPGs are in common use in proteomics investigations, not only along classical protocols but also for sample prefractionation and in shotgun approaches. Much less frequently are they used for 1-DE analytical applications, a field for which in recent years much attention has instead more often focused on capillary electrophoresis/IEF procedures.

2-DE with IPGs.

In order to overcome the limitations of carrier ampholyte generated pH gradients, IPGs were developed in the late 1970s. However, the 2-DE pattern we included in the first publication on IEF with IPGs [Bjellqvist et al., J. Biochem. Biophys. Methods 1982, 6, 317-339] was far from being competitive to O'Farrell's high-resolution 2-DE with carrier ampholytes. Our 2-DE pattern in this article was, more or less, only a proof of principle. It was, however, the beginning of a long journey of stepwise improved 2-DE protocols we developed in our laboratory and summarized in the reviews published in Electrophoresis 1988, 9, 531-546 and in Electrophoresis 2000, 21, 1037-1053. Milestones were the design of the IPG strip, and the "reduction-alkylation equilibration protocol" of IPG strips after IEF for the efficient transfer of proteins from first to second dimension. The protocol of 2-DE with IPGs has been constantly refined, e.g. by the generation of tailor-made IPGs with different pH intervals from the acidic to the basic extremes (pH 2.5-12), and extended separation distances for improved resolution. In the present review, a historical outline from the technical difficulties encountered during the development of 2-DE with IPGs, to the establishment of the actual "standard protocol" will be given, as well as the modified procedures for the separation of very acidic, very alkaline, low-abundance and hydrophobic proteins, followed by a brief discussion of the advantages and technical challenges of gel-based proteomic technologies.

The pre-omics era: the early days of two-dimensional gels.

I present a personal view of the beginning of two-dimensional gels and unsanctioned proteomics. I was still a young graduate student in the early 1970s when I developed methods for two-dimensional gel electrophoresis that became widely used. Though the method gave us the capacity to do things that had never been done, the value of global enumeration of proteins was not appreciated, and we were still two decades away from the invention of the term proteomics. I describe a period of exploration where, by exercising our new capability, we conducted the first proteomic type expression experiments, and made unforeseen contributions to advances in biology. Detection of single-amino acid substitutions validated genetic selections in cultured cells, and revealed a regulatory system that maintains the accuracy of protein synthesis by assuring an unbiased supply of its substrates. We documented biologic control with a dynamic range >10(8) fold, and, in a surprising turn, we identified an approach that provided a major breakthrough in recombinant DNA technology, the ability to express cloned sequences in Escherichia coli. The challenge then and now is to use a capability for global analysis to produce new insights into fundamental aspects of biology and to drive substantive technical advances.

Back to the future: the human protein index (HPI) and the agenda for post-proteomic biology.

The effort to produce an index of all human proteins (the human protein index, or HPI) began twenty years ago, before the initiation of the human genome program. Because DNA sequencing technology is inherently simpler and more scalable than protein analytical technology, and because the finiteness of genomes invited a spirit of rapid conquest, the notion of genome sequencing has displaced that of protein databases in the minds of most molecular biologists for the last decade. However, now that the human genome sequence is nearing completion, a major realignment is under way that brings proteins back to the center of biological thinking. Using an influx of new and improved protein technologies--from mass spectrometry to re-engineered two-dimensional (2-D) gel systems, the original objectives of the HPI have been expanded and the time frame for its execution radically shortened. Several additional large scale technology efforts flowing from the HPI are also described.

From proteomics to genomics.

Presently, science is moving from genomics to proteomics in order to get insight into the functional network of gene expression. Actually however, proteomics is much older than genomics and dates back to the introduction of the two-dimensional gel electrophoresis technique (2-DE) independently by Klose and O'Farrell. Based on this approach almost all cellular proteins can be separated. New developments in mass spectrometry allowed identification of single spots in the 2-DE protein pattern, including the underlying genes. Joachim Klose has focused his pioneering 2-DE studies on mouse models with special emphasis on quantitative protein variants. According to him, proteins are living molecules exhibiting a characteristic protein phenotype.