Spot overlapping in two‐dimensional maps: A serious problem ignored for much too long

Abstract

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.

Is protein overlap in two‐dimensional gels a serious practical problem?

“…We agree that both in theory and practice more than a single protein will migrate to each location on a 2-D gel and this has been discussed in several publications [1][2][3].…”

“…The article on spot overlap in 2-D gels by Campostrini et al [1] should not go without comment because it contains oversights and some unjustified, but dogmatic claims. We believe that this paper unduly discredits the potential of 2-DE and we therefore submit this response to introduce some objective analytical science and stimulate rational discussion.…”

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).…”

Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

“…Proteins within pI range of 4-7 exhibited significant overlap, which is known to interfere with quantification and identification. 30 To alleviate this problem, medium range pI 4-7 IPG strips were employed to quantify and identify the bulk of differential protein spots. Using this strategy, it was possible to resolve proteins within pI 4-7 clearly, and the spot overlap problem was minimized, a critical first step for differential proteomics screens.…”