Quantitative aspects of gel-based proteomics

Ernesto Silva , AnnSofi Sandberg and Bettina Levänen.

Prof. Pier Giorgio Righetti, Polytechnic of Milano, Department of Chemistry, Milan, Italy
Dr. Sue Mason, Nonlinear Dynamics, U.K
Dr. Andreas Hallberg, Ludesi, Sweden

Two-dimensional electrophoresis (2DE) is a prominent separation method for complex proteomes. In spite of numerous technical improvements in recent years, many sources of variance still prevail in modern 2DE technology. To this end, we are interested in improving the quantitative aspects of 2DE, both in terms of experimental methodology and post-electrophoretic analysis of the resulting 2DE image maps. A large portion of the experimental variance can be attributed to variability in the protein load due to inefficiencies in IPG strip reswelling or protein transfer from the 1st to the 2nd dimension. This effect causes an unknown variation in the total amount of protein present in any given 2DE gel and drastically impairs the ability to quantitatively determine the biological response to disease or toxicant exposure. To this end, we are constantly working on solutions to improve existing tools to correct for this type of variance, as well as developing new ones including development of novel fluorescent protein stains and internal standards. Another major research effort in our laboratory concerns post-electrophoretic aspects of quantitative 2DE, particularly in terms of the importance of various background subtraction, spot detection, and matching algorithms of 2DE analysis software packages as a source of variance.

Last updated: 2008-03-23