AC2DGel: analysis and comparison of 2D gels

Two-dimensional gel electrophoresis can retrieve information regarding thousands of different proteins from a crude protein sample. One of the major challenges in field of proteomics is to extract maximum information from 2D gels. In this study, we developed a web server for the analysis and comparison of 2D gels, which consists of three major modules. The first module allows the analysis of gels on the basis of molecular weight and pH. This module assists in calculating molecular weight and pH of a protein by clicking on corresponding spot at 2D gel image. The second module allows the comparison of two gels and presents the result as a superimposed image where spots/proteins on two gels can be examined. The useful feature of this module is that it allows the comparison of whole gel images or user specified areas or spots of gels. Besides this, it also allows zooming and other image transformations such as brightness and contract enhancement. The third module is an interface to the database of 2-D gel images maintained locally. The database consists of information about more than 3500 well annotated 2-D gel images obtained from public databases and literature. The server allows searching of gels from the database by keyword. Web server AC2Dgel is available for public from http://www1.imtech.res.in/raghava/ac2dgel/

Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation

Abstract BACKGROUND: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation. METHODS: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p<0.05, Anova). RESULTS: No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC>SU and 1 was SU>DC. Proteins present in 3 of the differential spots could be identified. One DC>SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC>SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU>DC spot contained pyruvate kinase M1/M2. CONCLUSIONS: The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential

Image Analysis Workflow for 2-D Electrophoresis Gels Based on ImageJ

A number of commercial software packages are currently available to perform digital two-dimensional electrophoresis (2D-GE) gel analysis. However, both the high cost of the commercial packages and the unavailability of a standard data analysis workflow, have prompted several groups to develop freeware systems to perform certain steps of gel analysis. Unfortunately, to the best of our knowledge none of them offer a package that performs all the steps envisaged in a 2D-GE gel analysis. Here we describe an ImageJ-based procedure, able to manage all the steps of a 2D-GE gel analysis. ImageJ is a free available image processing and analysis application developed by National Institutes of Health (NIH) and widely used in different life sciences fields as medical imaging, microscopy, western blotting and PAGE. Nevertheless no one has yet developed a procedure enabled to compare spots on 2D-GE gels. We collected all used ImageJ tools in a plug-in that allows us to perform the whole 2D-GE analysis. To test it, we performed a set of 2D-GE experiments on plasma samples from 9 patients victims of acute myocardial infarction and 8 controls, and we compared the results obtained by our procedure to those obtained using a widely diffuse commercial package, finding similar performance

Stochastic modeling for the COMET-assay

We present a stochastic model for single cell gel electrophoresis (COMET-assay) data. Essential is the use of point process structures, renewal theory and reduction to intensity histograms for further data analysis

Two-dimensional gel electrophoresis in proteomics: A tutorial

Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2)

Band-collision gel electrophoresis.

Electrophoretic mobility shift assays are widely used in gel electrophoresis to study binding interactions between different molecular species loaded into the same well. However, shift assays can access only a subset of reaction possibilities that could be otherwise seen if separate bands of reagent species might instead be collisionally reacted. Here, we adapt gel electrophoresis by fabricating two or more wells in the same lane, loading these wells with different reagent species, and applying an electric field, thereby producing collisional reactions between propagating pulse-like bands of these species, which we image optically. For certain pairs of anionic and cationic dyes, propagating bands pass through each other unperturbed; yet, for other pairs, we observe complexing and precipitation reactions, indicating strong attractive interactions. We generalize this band-collision gel electrophoresis (BCGE) approach to other reaction types, including acid-base, ligand exchange, and redox, as well as to colloidal species in passivated large-pore gels

Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry

Protein detection methods after electrophoresis have to be sensitive, homogeneous, and not to impair downstream analysis of proteins by MS. Speed, low cost, and user friendliness are also favored features. Silver staining combines many of these features, but its compatibility with MS is limited. We describe here, a new variant of silver staining that is completely formaldehyde-free. Reducing sugars in alkaline borate buffer are used as developers. While keeping the benefits of silver staining, this method is shown to afford a much better performance in terms of compatibility with MS, both in PMF by MALDI and in LC/ESI/MS/MS

The 68,000-Dalton Neurofilament-Associated Polypeptide is a Component of Nonneuronal Cells and of Skeletal Myofibrils

Purified preparations of 10-nm neurofilaments from rat spinal cord and bovine or porcine brain contain a predominant 68,000-dalton polypeptide. This polypeptide is also a major component of the neurofilaments that copurify with brain tubulin isolated by cycles of polymerization and depolymerization. A protein that has the same isoelectric point and molecular weight as the neurofilament-associated polypeptide has also been identified as a cytoskeletal protein in a variety of avian and mammalian cell types, including baby hamster kidney (BHK-21) mouse 3T3, Novikoff rat hepatoma, chicken fibroblast, and chicken muscle cells. This protein is also a component of isolated chicken skeletal myofibrils. One-dimensional peptide maps of the 68,000-dalton proteins purified by two-dimensional isoelectric focusing/NaDodSO4/polyacrylamide gel electrophoresis from myofibrils, cycled tubulin, purified neurofilaments, and various cultured cell types were identical. In immunofluorescence this protein was associated with cytoplasmic intermediate filaments and myofibril Z discs. These results indicate that the neurofilament-associated polypeptide is a conserved protein that is present in many different cell types in addition to neuronal cells. Because some of these cells contain the major components of two other intermediate filament classes, desmin and vimentin, a given cell type may contain the subunits of at least three distinct intermediate filament types

Characterization of the aerosol produced by infrared femtosecond laser ablation of polyacrylamide gels for the sensitive inductively coupled plasma mass spectrometry detection of selenoproteins

A 2D high repetition rate femtosecondlaserablation strategy (2-mm wide lane) previously developed for the detection of selenoproteins in gel electrophoresis by inductively coupled plasma mass spectrometry was found to increase signal sensitivity by a factor of 40 compared to conventional nanosecond ablation (0.12-mm wide lane) [G. Ballihaut, F. Claverie, C. Pécheyran, S. Mounicou, R. Grimaud and R. Lobinski, Sensitive Detection of Selenoproteins in Gel Electrophoresis by High Repetition Rate FemtosecondLaserAblation-Inductively Coupled Plasma Mass Spectrometry, Anal. Chem. 79 (2007) 6874–6880]. Such improvement couldn't be explained solely by the difference of amount of material ablated, and then, was attributed to the aerosol properties. In order to validate this hypothesis, the characterization of the aerosolproduced by nanosecond and high repetition rate femtosecondlaserablation of polyacrylamidegels was investigated. Our 2D high repetition rate femtosecondlaserablation strategy of 2-mm wide lane was found to produce aerosols of similar particle size distribution compared to nanosecond laserablation of 0.12-mm wide lane, with 38% mass of particles < 1 µm. However, at high repetition rate, when the ablated surface was reduced, the particle size distribution was shifted toward thinner particle diameter (up to 77% for a 0.12-mm wide lane at 285 µm depth). Meanwhile, scanning electron microscopy was employed to visualize the morphology of the aerosol. In the case of larger ablation, the fine particles ejected from the sample were found to form agglomerates due to higher ablation rate and then higher collision probability. Additionally, investigations of the plasma temperature changes during the ablation demonstrated that the introduction of such amount of polyacrylamidegel particles had very limited impact on the ICP source (ΔT~ 25 ± 5 K). This suggests that the cohesion forces between the thin particles composing these large aggregates were weak enough to have negligible impact on the ICPMS detection