عنوان مقاله
الکتروفورز ژل دو-بعدی در پروتئومیکس: گذشته، حال و آینده
فهرست مطالب
چکیده
الکتروفورز دو-بعدی و پیدایش پروتئومیکس
برداشتن پروتئومیکس
رسیدن به محدوده های الکتروفورز دو بعدی
استفاده از قدرت ژل های دو بعدی در تحقیقاتproteomic مدرن
نتیجه گیری
بخشی از مقاله
رسیدن به محدوده های الکتروفورز دو بعدی و ایجاد روشهای دیگر
بخاطر این تلاش گسترده با استفاده از الکتروفورز دو بعدی و طیف سنج جرمی بعنوان ابزار اصلی، داده های بسیاری جمع آوری شدند و مورد تحلیل قرار گرفتند و بزودی مشخص شد که همیشه همان نوع پروتئین های یکسان بود که پیدا می شود و همان نوع پروتئین های یکسان بود که وجود نداشت. برای بهبود بخشیدن به تفکیک پروتئین های آب گریز، تلاش های زیادی انجام شد تا واگشاپذیری پروتئین در موقعیت های متداول در بعدIEF بهبود یابد. و این شامل تغییراتی درchaotropes استفاده شده درIEF و همچنین در شوینده های استفاده شده برای این مرحله بود.
کلمات کلیدی:
From mute star maps to protein buzz: two-dimensional electrophoresis and the birth of proteomics SDS electrophoresis in its modern form was introduced in the early 70s [1] and soon became very widely used by protein biochemists. However, analysis of complex cellular extracts by this method made obvious the fact that the resolution was far from being sufficient to separate the many protein components of such extracts. To increase the resolution to a sufficient extent, it was necessary to couple two independent separations. At that time (and this is still the case) it was obvious that the separation best able to complement SDS electrophoresis was isoelectric focusing, and this was the coupling that researchers in protein separation tried to achieve. One of the first reports of successful two-dimensional electrophoresis coupling denaturing IEF to SDS PAGE was published in 1974 [2] but went relatively unnoticed by the community, probably because of two features. The first one was the inclusion of the sample within the IEF tube gel, which is a quite efficient but slightly cumbersome process. The second was the detection method chosen, i.e. the classical Coomassie blue staining in alcohol–acid mixtures. The poor sensitivity of the method led to nice but rather “empty” 2D images, and therefore not that impressive. The situation changed dramatically the year after, with the seminal paper of O'Farrell [3]. This paper coupled better technical choices (loading of samples on top of IEF gels), a very detailed description of the protocol, and detection by autoradiography, showing at once hundreds of protein spots on a single gel. Many researchers got very impressed by the results and underlying power of the method, so that 2D electrophoresis spread quite widely and quickly around the world. This is clearly shown by the citation index of the paper, gathering more than 1500 citations from 1976 to 1980. As soon as that time (late 70s very early 80s), the first applications of what was to become proteomics took off, with applications to cell biology [4,5] and clinical biology [6], with the first ancestor of the human proteome project [7]. However, it must absolutely be stressed that protein identification at that time was quite an ordeal. The main methods were comigration with purified proteins [8] and blotting with antibodies [9,10]. Thus, it was possible to say where was protein X (purified or with an available antibody) on the gels, but it was very difficult to know who was spot Y. For example, it took several years to know that cyclin/PCNA, identified as a cell cycle dependent protein [11] was indeed a subunit of DNA polymerase delta [12]. In the absence of readily-available protein identification tools, lot of attention was brought to the use of quantitative protein patterns as identifiers of cellular states via correlation analyses. It is often forgotten that statistical analysis in proteomics did not start a few years ago by getting inspired from transcriptomics, but long before the word proteomics was even coined, and in fact some 20–25 years ago. Indeed, computer analysis included both gel image analysis [13–16], but also strong data analysis with multivariate methods [17–22]. However, it was not by chance that one of the first 2D gel analysis system was named TYCHO [14], after the name of the famous astronomer (and alchemist) Tycho Brahe, as 2D maps were much like silent star maps for more than ten years (1977 to 1987). In addition to this dearly-resented lack of universal protein identification system, 2D electrophoresis was plagued at that time with major reproducibility problems, and was much more an art than anything else. New practitioners of the field may not realise that the IEF gels were soft unsupported tube gels (typically 4% acrylamide) that were to be extruded from the glass tube containing them for the first dimension in order to be loaded on the second dimension slab gel. This was quite a difficult process, and a lot of non-linear deformation of the tube gel took place there. Ending with lengths variations of 10– 20% was not uncommon, and this was highly detrimental to spot positional reproducibility. Moreover, IEF with carrier ampholytes is by itself a process that must be carefully controlled to achieve reproducibility. Carrier ampholytesbased pH gradients are not really stable, and prone to cathodic drift, i.e. progressive loss of the basic portion of the gradient, so that the migration profile (in Volts and Volt hours) had to be carefully controlled. Moreover, the fine profile of the pH gradient is dependent on the precise composition of the carrier ampholytes, and this varies from batch to batch, leading to long-term irreproducibility of the IEF separations. Despite these difficulties, exquisite experiments could be devised, as the demonstration of the expression of several actin forms in a single cell [23]. In addition, important dimensions of our current proteomic knowledge root from that period, such as the number of proteins present in a mammalian cell [24] or the knowledge that serum proteins are extensively modified [8]. The best was to come in the last 80s, with two key events for the development of two-dimensional electrophoresis in biology. The first key event was the introduction of immobilized pH gradients in the first dimension. By construction, immobilized pH gradients eliminate the reproducibility problems associated with carrier ampholytes-based pH gradients. However, initial attempts to adapt them for 2D electrophoresis, either in the tube gel mode [25] or via a gel slab [26] were not very successful, and the final solution, i.e. the now classical gel strips, was introduced later [27]. Further work was necessary to ensure good interfacing of the IPG strips with the SDS slab gel [28], but a stable protocol was soon available for the community [29,30]. With the establishment of IPGs, 2D electrophoresis ceased to be a difficult art to convert only into good craftsmanship. There was a real quantum leap in reproducibility, going up to interlaboratory studies with a positive outcome [31,32], something that was just unbelievable a few years before. The real change of paradigm, however, came with the longawaited interface of 2D gels with protein microanalysis techniques. This was some years before mass spectrometry entered the scene, and obtained through the progress of Edman sequencing. The late 80s saw a blossoming of microsequencing techniques applied to gel-separated proteins, e.g. directly N-terminal sequencing [33], easy to perform and sensitive (only 1–2pmol required) but requiring a free Nterminus, something not often available in eukaryotic proteins. To bypass this limitation, internal sequencing protocols were developed, either from blots [34,35] or directly from
