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Microscale solution IEF combined with 2‐D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts
Author(s) -
Han MeeJung,
Herlyn Meenhard,
Fisher Aron B.,
Speicher David W.
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700337
Subject(s) - proteome , difference gel electrophoresis , microscale chemistry , chemistry , chromatography , fractionation , proteomics , biochemistry , mathematics education , mathematics , gene
Current gel‐based protein profiling methods such as 2‐DE and fluorescent 2‐D difference in gel electrophoresis (DIGE) evaluate small portions of complex proteomes. Hence, sample prefractionation is essential for more comprehensive proteome coverage and detection of low‐abundant proteins. In this study, we describe the combination of DIGE labeling with microscale solution IEF (MicroSol‐IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2‐D gels. By fluorescently tagging and mixing samples and controls prior to prefractionation, complications resulting from minor run‐to‐run variations during MicroSol‐IEF separations of multiple samples are avoided. This greatly improves the reliability of quantitative comparisons. To illustrate its utility, this 3‐D DIGE strategy was applied to analysis of human melanoma cells and mouse lung tissue extracts. Approximately 1000 reproducible spots can be obtained from narrow range 2‐D gels of individual MicroSol‐IEF fractions, and approximately 6000 spots can be obtained from entire proteomes. Quantitative changes in closely related samples could be more reliably detected and the method has a greatly increased capacity to distinguish between closely related protein isoforms. Thus the 3‐D DIGE strategy produces a powerful method for more comprehensive and more reliable quantitative comparisons of protein profiles of very complex proteomes.