Real and imaginary artefacts in proteome analysis via two-dimensional maps

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Pier Giorgio Righetti

Abstract

The present review touches on a long-lasting debate on possible artefacts (i.e. generation of spurious spots, not belonging to the biological sample under analysis) induced by the separation technique (in this case, two-dimensional mapping) per se. It is shown here that some of the biggest offenders, always blamed in the past (at least since 1970, i.e. since the inception of gel-base isoelectric focusing protocols), namely deamidation (of Asn and Gln residues) and carbamylation (due to cyanate produced in urea solution), simply do not occur in properly handled samples and have never indeed been demonstrated in real samples, except when forced in purpose. Conversely, two unexpected major artefacts have been recently shown to plague 2D mapping. One is formation of homo- and hetero-oligomers in samples that have been reduced but not alkylated prior to entering the electric field. The phenomenon is highly aggravated in alkaline pH regions and can lead to an impressive number of spurious spots not existing in the original sample. Thus, alkylation (best if performed with acrylamide or vinylpyridines) is a must for avoiding such spurious spots, as well as sample streaking and smearing in the alkaline gel region, and for maintaining sa...Continue Reading

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Citations

Jul 24, 2012·Amino Acids·Ping-Chung ChenJunmin Peng
Sep 10, 2014·International Journal of Molecular Sciences·Susana CorreiaGilberto Igrejas
Jan 20, 2015·Proteomics. Clinical Applications·Thomas A Gorr, Johannes Vogel
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Jan 22, 2013·Proteomics·Laxmikanth Kollipara, René P Zahedi
Mar 1, 2014·Biotechnology and Applied Biochemistry·Neda Saraygord-AfshariMostafa Naderi
Jan 10, 2013·Current Opinion in Chemical Biology·Kathrine B SylvestersenMichael L Nielsen
Jul 4, 2015·Journal of Agricultural and Food Chemistry·Marcella Michela GiulianiZina Flagella
Dec 20, 2014·Analytical Chemistry·Elisa FasoliAlfonsina D'Amato
Jun 15, 2011·Journal of Proteomics·Thierry Rabilloud, Cécile Lelong
Feb 19, 2013·Journal of Agricultural and Food Chemistry·Marianna PompaZina Flagella
Feb 8, 2018·Analytical and Bioanalytical Chemistry·Trina Mouchahoir, John E Schiel
Jun 8, 2007·Expert Review of Proteomics·Stefano BarelliJean-Daniel Tissot
Sep 13, 2012·Expert Review of Proteomics·Michela Di MicheleKathleen Freson
Mar 23, 2011·Electrophoresis·Erhua ZhangXuanqiang Liang
Sep 29, 2012·Electrophoresis·Jochen HeukeshovenGenrich V Tolstonog
Dec 8, 2016·Electrophoresis·Stuart Brown, Gillian Norris
May 17, 2012·Electrophoresis·Patrick W ReedRobert J Bloch
Nov 8, 2013·Journal of Proteome Research·Benjamin B A RaymondSteven P Djordjevic

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