The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high-throughput methods has led us to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.

Proteome and proteomics: New technologies, new concepts, and new words

The two-dimensional electrophoresis (2-DE) technique developed by Klose in 1975 (Humangenetik 1975, 26, 211-234), independently of the technique developed by O'Farrell (J. Biol. Chem. 1975, 250, 4007-4021), has been revised in our laboratory and an updated protocol is presented. This protocol is the result of our experience in using this method since its introduction. Many modifications and suggestions found in the literature were also tested and then integrated into our original method if advantageous. Gel and buffer composition, size of gels, use of stacking gels or not, necessity of isoelectric focusing (IEF) gel incubation, freezing of IEF gels or immediate use, carrier ampholytes versus Immobilines, regulation of electric current, conditions for staining and drying the gels - these and other problems were the subject of our concern. Among the technical details and special equipment which constitute our 2-DE method presented here, a few features are of particular significance: (i) sample loading onto the acid side of the IEF gel with the result that both acidic and basic proteins are well resolved in the same gel; (ii) use of large (46 x 30 cm) gels to achieve high resolution, but without the need of unusually large, flat gel equipment; (iii) preparation of ready-made gel solutions which can be stored frozen, a prerequisite, among others, for high reproducibility. Using the 2-DE method described we demonstrate that protein patterns revealing more than 10 000 polypeptide spots can be obtained from mouse tissues. This is by far the highest resolution so far reported in the literature for 2-DE of complex protein mixtures. The 2-DE patterns were of high quality with regard to spot shape and background. The reproducibility of the protein patterns is demonstrated and shown to be thoroughly satisfactory. An example is given to show how effectively 2-DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross (Mus musculus x Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structural analysis of the human genome, currently pursued most intensively on a worldwide scale, should be accompanied by a functional analysis of the genome that starts from the proteins of the organism.

Two‐dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome

https://hal.archives-ouvertes.fr/hal-00509715/document

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

Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining

Multiple spontaneous transient elevations of cytosolic-free calcium ([Ca2+]i) are observed in single human neutrophils during adherence. The interrelation between adherence and spontaneous [Ca2+]i transients was analyzed by simultaneous monitoring of [Ca2+]i and cell morphology. Fluorescent images of fura 2-loaded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]i was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2+]i transients were abolished by chelation of extracellular Ca2+. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]i activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of [Ca2+]i transients. To determine if the movement alone or adhesion alone is responsible for [Ca2+]i activity, we treated cells with antibodies against the beta chain (CD18, beta 2) or the alpha subunit (CD11b, alpha m) of the dominant leukocyte integrin (CR3). Antibody-treated cells showed normal extension of pseudopods but impaired ability to adhere. Inhibition of adhesion in this way inhibited [Ca2+]i activity. Taken together these results suggest that following sequence of events after contact of neutrophils with surfaces: (a) cell movement and shape change lead to enhanced contact of integrins with the surface; and (b) integrins-mediated adhesion generates multiple [Ca2+]i transients. The [Ca2+]i transients may then control exocytic events associated with movement and may provide a link between adherence and activation or priming of neutrophils to other stimuli.

https://rupress.org/jcb/article-pdf/112/6/1249/1060979/1249.pdf

Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family.

The interpretation of two-dimensional gel electrophoresis (2-DGE) profiles can be facilitated by artificial intelligence and machine learning programs. We have incorporated into our 2-DGE computer analysis system (termed MELANIE-Medical Electrophoresis Analysis Interactive Expert system) a program which automatically classifies 2-DGE patterns using heuristic clustering analysis. This program is a step toward machine learning. In this publication, we describe the classification method and the preliminary results obtained with liver biopsy electrophoretograms. Heuristic clustering is also compared to other classification techniques.

Automatic classification of two-dimensional gel electrophoresis pictures by heuristic clustering analysis: a step toward machine learning.

https://archive-ouverte.unige.ch/unige:47497/ATTACHMENT01

Christian Maurice Roch

Papers

Automatic classification of two-dimensional gel electrophoresis pictures by heuristic clustering analysis: a step toward machine learning.

Automatic learning strategies and their application to electrophoresis analysis.