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J Biomol Tech. 2007 February; 18(1): 75.
PMCID: PMC2291894

P216-T Beyond Differential In-Gel Electrophoresis: Top-down Multiplexed Analysis in Two-dimensional Gels Using an Isobaric Mass Tagging Approach

Abstract

Large sets of biological samples are commonly encountered in biomedical research, and a rigorous, reliable top-down quantification method for proteins from multiple samples is required. With differential in-gel electrophoresis (DIGE), Cy3 and Cy5 dyes are used to fluorescently label two different protein samples prior to running them on the same 2D gel. Often, Cy2 dye is used as an internal standard as well. This approach permits analysis of two to three samples under identical conditions, eliminating the need to register and match the images as in traditional 2D gels. Though conceptually seductive, problems plague DIGE, especially when attempting to perform complex quantitative proteomics studies. Since the DIGE images are generated with two to three different dyes of differing molar extinction coefficients, quantum yields, and physicochemical properties, absolute quantification of small intensity differences is challenging. Also, DIGE is overly simplistic in its basic assumption that all information required in an experiment can be obtained from a single 2D gel using an easy control-vs.-perturbed state experimental design. Multi-point time-course experiments and drug dose-response experiments require many more than the two to three sample capacity of DIGE. An isobaric mass tagging strategy is presented wherein seven different samples are simultaneously fractionated on a single 2D gel and protein abundances subsequently quantified by mass spectrometry. Workflows that replace or alternatively augment DIGE are described. Reproducibility of conventional DIGE vs. the isobaric mass tagging strategy demonstrates higher accuracy quantification obtained with the latter approach.


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