Microscale solution IEF combined with 2-D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts.
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.
Han
Mee-Jung
MJ
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering, BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea.
Herlyn
Meenhard
M
Fisher
Aron B
AB
Speicher
David W
DW
D000818:Animals; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D008168:Lung / Q000737:chemistry; D008545:Melanoma / Q000737:chemistry; D051379:Mice; D008819:Mice, Nude; D008832:Microchemistry / Q000379:methods; D020543:Proteome / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods
D020543:Proteome