Workshops

Proteomics research tools are developed to separate, characterize, and catalog proteins of interest or all proteins in a biologic sample. In order to achieve this the proteins in a tissue, cellular system or body fluids are first separated from the accompanying biologic molecules, such as DNA, lipids, carbohydrates as well as salts, and other small molecules. In subsequent separation steps, proteins are separated from each other to allow a mass spectrometry-based identification. In this presentation, we will discuss the various methods for protein separation and how multiple steps can be combined for a deeper coverage of the proteome. Although 2D gel electrophoresis with its orthogonal mechanism of charge and molecular size separation can provide resolution of up to several thousand proteins on a single gel, both fractionation and pre-concentration of proteins are necessary because orders of magnitude, more proteins can be present. We are presenting strategies and data using both chromatographic and electrophoretic methods in two biomarker discovery programs with serum and brain samples. In the case of serum, where sample volumes of 10 ml can be obtained, the dominant proteins albumin and IgGs are chromatographically removed via two affinity columns before the remaining proteins are fractionated with the Rotofor and separated on microrange 2D gels. This approach allows almost tripling the amount of detected proteins thus increasing the chance of finding a biomarker. Mouse and rat brain samples provide smaller total amount of proteins. After fractionation based on ionexchange on a microspin column, further fractionation was achieved with the micro-Rotofor, which is capable of handling sample volumes of as little as 2.5 ml before 2D gel separation and MS identification. Finally with the Experion, a chip-based, microliter volume, fast and automated protein sizing is possible. We explore the use of the Experion in quickly assessing which fraction holds differentially expressed proteins thus cutting the number of gels to be run and time on MS for protein ID.