Gel Electrophoresis-based Proteome Research Strategies And Applications

With the completely sequencing of human genome, a new era -post-genome era is coming. The key task of life science transferred from large scale sequencing of genome to the functional characterization of genes. Proteome has become one of the hot fields in life sciences. The necessities for large scale of proteome research are separation techniques with high resolution and proteins identification techniques with high throughput and high sensitivity. So in the first part of this research, 2-DE separations using alkaline IPG gel and ultra-zoom gel in the IEF dimensional were optimized. Using the optimized parameters, a high resolution 2-DE profiling of fetal liver total protein with 5487 protein spots was presented. At the same time, techniques for high throughput identification of in-gel protein from 2-DE gels (in the first part of the paper) and SDS-PAGE gels (in the second part of the paper) were established and optimized. Human fetal liver in 4-6 months of gestation performs physiological functions of normal liver as well as embryonic hematopoiesis and immune development. A large scale of proteome analysis of fetal liver, may contribute to the discovering of functional proteins associated with proliferation, hematopoiesis and development. Using sub-proteome technique combined with three types of gel separation and multi-stage mass spectrometries, 1045 non-redundant fetal proteins were measured, including 902 functionally annotated proteins and 143 new proteins, which constituted a proteome database of fetal liver. Functional categories and relative abundance analysis of fetal liver proteins were performed. A number of proteins specifically related with normal liver, liver cancer, hematopoiesis and development were inferred after comparisons between fetal liver proteome database and nine other proteome databases.An important application of proteomics is the probe of potential tumor markers. In second part of this research, proteins secreted during culture of normal and tumoral bronchial epithelial cells in condition medium (CM) were measured using SDS-PAGE and nano-ESI-MS/MS. 238 non-redundant proteins were identified from 4 pairs of matched CM and 1 tumor CM. Comparisons with known serum and urinary protein dabasets showed that 153 proteins were reported for the first time as secreted proteins in this research. All of the 9 proteins selected for further ELISA test in serum from clinical patients gave positive results. Most of the proteins detected differentially between normal and cancer CM showed similar trends between normal and cancer serum, which inferred these proteins could be potential tumor markers.In conclusion, firstly, proteome database of human fetal liver was established, as we could know, it is the most comprehensive proteome database of human organ up to now. Secondly, a totally new strategy for tumor proteome research was put forward. The strategy is very effective in enriching low abundant secretory proteins and avoiding disturbances from high abundant protein in body fluid. It will be proved to be a high throughput biomarker discovery platform in tumor research as well as in other key diseases.