The Fluorescence Differential Gel Electrophoresis Technique For The Establishment Of Its Application Of Proteomics Research In The The Epidermis Difference In The Keratinocyte-specific Smad4 Knockout Mice

Smad4 is the common mediator of transforming growth factor-B (TGF-B) superfamily signaling, which functions in diverse developmental processes in mammals. To study the role of Smad4 in skin development, a keratinocyte-specific null mutant of Smad4 (Smad4^co/co; K5-Cre) in mice has been successfully established using the Cre-loxP system in YANG X'lab. The Smad4-mutant mice exhibited progressive alopecia as a result of the mutant hair follicles failing to undergo programmed regression. Seventy percent of Smad4^co/co; K5-Cre mice developed spontaneous tumors (including skin tumor) within 12 months of birth.A cell is normally dependent upon a multitude of metabolic and regulatory pathways for its survival, and proteins are the active agents in those pathways. Proteomics, a newly emerging realm which aims at the large-scale study of protein expression and regulation, has grown at an astonishing rate, and shows no sign of slowing. Comparative proteomics was applied to an enormous variety of biological questions through measuring changes in protein expression between samples. The proteomics researcher now has access to a powerful set of techniques for proteome study, including 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification) and so on. In spite of its succeeding high resolution of traditional 2-DE, highl reproducibility, high throughput and widely dynamic range, 2D DIGE has been becoming a sophisticated technique for the study of differential proteomicsIn order to obtain proteins related with Smad4 signal pathway at a high throughput and large scale level, and then investigate molecular mechanisms in alopecia and skin tumor formation, the technique of 2D DIGE has been developed and applied to analyze differentially expressed proteins between Smad4^-/- and Smad4^co/co; K5-Cre. The protocol for sample preparation in mice epidermis tissue was optimized and the sensitivity and reproducibility of 2D DIGE was evaluated, at the same time ratio thresholds about the sample were estimated. We identified 241 differentially expressed spots, 167 of them were identified by tandem mass spectrotetry with high confidence. These proteins involving 14-3-38, Galectin-7, FABP5, cathepsin D and Enolase 1 etc. A great deal of bioinformatics analysis about them was performed. Some of them were further validated by Western blotting.