Power Of Two-dimensional Gel Electrophoresis Research Articles

A Proteomic Analysis of Human Follicular Fluid: Comparison between Younger and Older Women with Normal FSH Levels

The follicular fluid (FF) is produced during folliculogenesis and contains a variety of proteins that play important roles in follicle development and oocyte maturation. Age-related infertility is usually considered as a problem that can be solved by assisted reproduction technology. Therefore, the identification of novel biomarkers that are linked to reproductive aging is the subject of this study. FF was obtained from healthy younger (20–32 years old) and older (38–42 years old) women undergoing intracytoplasmic sperm injection (ICSI) due to male factor infertility. The FF was analyzed by two-dimensional gel electrophoresis (2-DE). The power of two-dimensional gel electrophoresis and the identification of proteins were exploited using matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI-TOF-TOF) mass spectrometry. Twenty three protein spots showed reproducible and significant changes in the aged compared to the young group. Of these, 19 protein spots could be identified using MALDI-TOF-TOF-MS. As a result of MASCOT search, five unique downregulated proteins were identified in the older group. These were identified as serotransferrin, hemopexin precursor, complement C3, C4 and kininogen. A number of protein markers were found that may help develop diagnostic methods of infertility.

Plasma proteomics analysis of tamoxifen resistance in breast cancer

Due to ease accessibility, exploring plasma for candidate cancer biomarkers is of great interest to molecular biologist and physicians. In breast cancer, the development of tamoxifen resistance (TR) is among the major causes of recurrence and mortality. Therefore, the identification of novel biomarkers that are linked to TR is of great interest and the subject of intensive research. Here, we exploited the power of two-dimensional gel electrophoresis coupled with protein identification using tandem mass spectrometry to identify plasma proteome signatures associated with TR. Comparative proteomics analysis resulted in the identification of 15 statistically significant spots, which were up-/downregulated after tamoxifen therapy. MASCOT search of the mass spectrometry generated spectral data resulted in the identification of 9 proteins. Several differentially expressed proteins such as clusterin, serum amyloid A, serpin B4, and transthyretin are already known to be involved in cancer incidence and progression. The possible involvement of these candidate proteins in conferring TR and their potential usefulness as plasma biomarkers for predicting response to tamoxifen treatment has been discussed.

Proteomic Analysis of Putative Latex Allergens

Background: Extensive analysis of allergenic proteins is generally time-consuming and labor-intensive. Accordingly, a rapid and easy procedure for allergen identification is required. As sequence information on proteins and genes is accumulated in databases, it is becoming easier to identify a candidate protein using proteomic strategies, i.e. two-dimensional gel electrophoresis, site-specific fragmentation, mass spectrometry and then database search. In this study, we evaluated the usefulness of a proteomic strategy for identifying putative allergens through its application to latex proteins. Methods: Latex proteins were separated with two-dimensional gel electrophoresis, and putative allergens were visualized by IgE immunoblotting using pooled serum from latex-sensitive patients. The IgE-interactive proteins were cut out from the negatively stained two-dimensional gel and subjected to in-gel digestion by trypsin. Then the resulting peptides were analyzed with mass spectrometry. Based on the mass spectrometric data we obtained, the allergen candidates were assigned by a database search. Results: Five previously reported allergens and five new allergen candidates were identified with the proteomic approach without isolating the individual proteins. Less than 1 mg of crude latex protein was sufficient for the entire protocol. Because plural proteins can be processed in parallel, analysis of about 50 IgE-interactive proteins was accomplished within 1 week. Conclusions: Analysis of putative allergens with proteomic strategies (allergenomics) is a promising avenue for rapid and exhaustive research. The high resolving power of two-dimensional gel electrophoresis is superior to conventional gel electrophoresis. Moreover, the notable sensitivity and speed of mass spectrometry have pronounced advantages over the N-terminal sequencing that has generally been used for protein identification.

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

To control the G1/S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27kip1 and p21cip1) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15- or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.

Informatic Tools for Proteome Profiling

In recent years, the practice of proteomics research has experienced a dramatic shift within the pharmaceutical and biotechnology industry with the widespread implementation of novel applications. The areas of interest extend all the way from discovery of novel drug, vaccine, and diagnostic targets, characterization of protein-based products, toxicology, and identification of surrogate markers of activity in clinical research, to the ability to provide information on the mechanisms of drug action. The power of two-dimensional gel electrophoresis as well as advances in mass spectrometric techniques combined with sequence database correlation have enabled speed and accuracy in identification of proteins in complex mixtures. This article surveys currently available software and informatic tools related to these methods for proteome profiling. The broad acceptance of these technologies, however, has not been accompanied by significant advances in the informatics and software tools necessary to support the analysis and management of the massive amounts of data generated in the process. In this context, this article also discusses the importance of relational databases for protein identification data management.

Rapid electroelution of two-dimensionally separated protein mixtures: its use in in vitro assays of T cell activities.

A recently developed electroelution method for separated mixtures of proteins and its application in vaccine research were investigated. The method combines the high resolution power of two-dimensional gel electrophoresis with the advantage of direct probing of separated proteins with viable cells. An electroelution time of only 30 min was sufficient for complete protein transfer, as shown by Coomassie Brilliant Blue and silver staining. Inclusion of sodium dodecyl sulfate (SDS) into the electrophoresis buffer for the second dimension considerably improved the separation capacity. Furthermore, because of the low concentration of SDS (0.03%) no deleterious effects on the cells were seen. It was shown that T lymphocytes from cattle vaccinated with dead M. bovis BCG responded to numerous mycobacterial protein antigens, whereas unvaccinated control animals showed no, or very weak, responses. A comparison of T cell proliferation profiles obtained with different protein separations demonstrated the reproducibility of the method.

Sequential two-dimensional and acetic acid/urea/Triton X-100 gel electrophoresis of proteins

A method is described which combines the resolving power of two-dimensional gel electrophoresis with that of acetic acid/urea/Triton X-100 gel electrophoresis, avoiding the necessity of eluting protein from the gels at any step of the procedure. The combination of electrophoretic separation on the basis of charge, mass, and hydrophobic properties of the proteins has the potential of resolving modified forms and isoforms present in very complex protein populations. The technique can be used for analytical purposes, or it may be scaled up to yield microgram amounts of highly purified proteins. The resolution obtained by tandem application of nonequilibrium pH gradient electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyacrylamide gel electrophoresis in the presence of nonionic detergent was evaluated using crude nuclear proteins of the nematode Caenorhabditis elegans.

A double-label two-dimensional procedure for the analysis of membrane proteins.

A previously described double-label two-dimensional electrophoresis procedure (Wheeler et al., Anal. Biochem. 1986 159, 1-7) for the analysis of differences between two complex mixtures of soluble proteins has been modified to allow analysis of proteins requiring detergent for aqueous solubility. The samples are first disrupted by sonication and the insoluble proteins concentrated by high-speed centrifugation. The proteins are then solubilized with sodium dodecyl sulfate and further concentrated in a centrifugal concentrator to achieve protein mixtures suitable for labeling with 14C and 3H by reductive methylation and subsequent two-dimensional electrophoresis. The sample concentration step is quick, minimizes the concentration of sodium dodecyl sulfate in the final sample, and avoids the potential difficulties associated with lyophilization or precipitation. The modified procedure was applied to the analysis of erythrocyte membranes, platelets and isolated placental microvilli. The high resolving power of two-dimensional gel electrophoresis is retained and the procedure is sensitive because the conditions of labeling allow substantial incorporation of radioactivity into protein despite the presence of detergent.

Quantitative double-label radiography of two-dimensional protein gels using color negative film and computer analysis.

We have devised a method of data collection and computer analysis which allows utilization of the resolving power of two-dimensional gel electrophoresis of proteins, in conjunction with the versatility of using two different radionuclides simultaneously. Cultures of Escherichia coli growing with exponential growth rate constants (mu) of 0.32 and 1.43 were labeled with [3H]leucine and [14C]leucine, respectively; these samples were mixed, and cell protein was separated on a two-dimensional gel. Spacial and quantitative data for both radionuclides were recorded on color negative film by radiographic exposure. Data for 14C alone were then collected photographically from the red-light-sensitive layer of the film using a red filter, while data for 3H and spillover of 14C were collected photographically from the blue-light-sensitive layer using a blue filter. These two data sets were analyzed by CINT, a computer program for analysis of two-dimensional gels, and quantitative data for 3H were calculated after determination of spillover of 14C in a manner analogous to quantification of 3H and 14C by liquid scintillation counting. Quantitative data from over 1000 protein spots representing from 0.002% to 10% of the total 3H or 14C, respectively, are available in a matter of hours. We have used this method to analyze the effect of growth rate and medium composition on the relative levels of individual proteins in a pathogenic strain of E. coli which contains group 111 O-antigen. As expected, the relative levels of aminoacyl-tRNA synthetases, protein chain elongation factors, ribosomal proteins, and the alpha-subunit of RNA polymerase are all increased with increased growth rate; the magnitude of these changes agreed with previous data derived using other strains of E. coli. Alterations in the levels of other proteins identified on the two-dimensional gels could be interpreted in terms of changes in medium composition. When compared to manual data collection by excising radiolabeled proteins and quantifying 3H and 14C in a liquid scintillation counter following combustion to H2O and CO2, respectively, this new method of data collection and computer analysis increases the resolution of data collection and decreases the time involved from days to hours.