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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\u2009proteins 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 \u0394p<jats:italic>I<\/jats:italic> and \u0394<jats:italic>M<\/jats:italic><jats:sub>r<\/jats:sub> 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 <jats:italic>ca.<\/jats:italic> 1\u2005mg of total protein are applied and typically at least 1000\u2009spots 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\u2010dimensional map are discussed.<\/jats:p>","DOI":"10.1002\/pmic.200401253","type":"journal-article","created":{"date-parts":[[2005,5,6]],"date-time":"2005-05-06T23:49:47Z","timestamp":1115423387000},"page":"2385-2395","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":119,"title":["Spot overlapping in two\u2010dimensional maps: A serious problem ignored for much too long"],"prefix":"10.1002","volume":"5","author":[{"given":"Natascia","family":"Campostrini","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liliana B.","family":"Areces","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juri","family":"Rappsilber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Chiara","family":"Pietrogrande","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco","family":"Dondi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabio","family":"Pastorino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mirco","family":"Ponzoni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pier Giorgio","family":"Righetti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2005,6,2]]},"reference":[{"key":"e_1_2_1_2_2","volume-title":"Biological Chemistry","author":"Mahler H. 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