Spot overlapping in two‐dimensional polyacrylamide gel electrophoresis maps: Relevance to proteomics

Abstract

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.

Different protein profiles in inferior colliculus and cerebellum: A comparative proteomic study

“…The number of spots for which we identified two or more overlapping proteins (25%) fits with a published model which predicts the number of overlapping proteins (Pietrogrande et al, 2003;Campostrini et al, 2005). We are therefore confident that we indeed detected the majority of overlapping proteins.…”

“…An overlap of different proteins in a single spot represents a problem for two-dimensional gel electrophoresis-based quantification methods (Pietrogrande et al, 2003;Campostrini et al, 2005). The number of spots for which we identified two or more overlapping proteins (25%) fits with a published model which predicts the number of overlapping proteins (Pietrogrande et al, 2003;Campostrini et al, 2005).…”

Prefractionation techniques in proteome analysis: The mining tools of the third millennium

“…Nevertheless, some recent papers suggest that the problem of this vast complexity could be overcome with no fractionation but rather by running a series of narrow-range IPG strips (covering no more than 1 pH unit). Hoving et al [5] and Westbrook et al [6] note that "zoom" and "ultrazoom" gels are quite important for avoiding or at least minimizing the problem of spot overlapping in 2-DE, an ever present hazard in 2-DE maps [7][8][9]. The use of large-size gel slabs (18 cm or longer in the first dimension, 18620 cm, or larger, in the second dimension), would even dramatically increase the resolution, as reported by Corthals et al [10] and Wildgruber et al [11].…”

Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

“…To achieve better separation, a relatively complicated procedure based on 2-D PAGE has been used for the separation of these enzymes in the extracting cytosol from rat hepatocytes [2], based on the fact that the enzymes coexist with many other proteins in the cited hepatocytes. However, 2-D PAGE requires higher protein concentrations or higher loading sample volumes when the proteins are detected with the CBB-R250 staining method, the experimental operation being tedious [14,15]. Furthermore, the lack of a highly selective staining method especially for the detection of the bands of the enzymes from complicated matrices limits its application.…”