The Sumo Modification Of The Size Of The Protein Identification And The Establishment Of The Verification Techniques Of Protein Sumoylation

Post-translational modifications (PTMs) is a very important regulatory mechanism on the level of post-translation.They helps organisms efficiently and rapidly respond to the extracellular stimuli by modifying the protein and subtily regulating the protein function.Ubquitin and Ubiquitin-like proteins(Ubls) are important kinds of PTMs, they modify the substrate proteins on one or one more sites. SUMO(small ubiquitin-like modifier), one kind of Ulps, modify the substrate protein by form the colavent bond between the C-terminal of them and the lysine of substrate. Many studies show that sumoylation involed in many cellular biological processes including cell cycle regulation, signal tranduction, mitochondrial fission, mRNA trafficking, DNA damage repair, regulation of genomic stability and so on.Many target proteins which can be modified by SUMO have been found by using the proteomic and bioinfomatic prediction methods. However, the proteomic method used before mainly enriched and identified the sumoylated proteins which modified by overexpressed exogenous SUMO by affinity chromatography, it can not present the real sumoylation state in cell. So a new method is needing for enriching and identifying the endogenous sumoylated proteins.The thesis described the establishment of a new poteomic method for large scale purification and identification the proteins modified by endogenous SUMO. We first prepared and identified the anti-human SUMO1 and SUM02 monoclonal antibodies. Then we prepared the antibody affinity column by crosslinking the anti-SUMO1 antiboy with the Protein A-agarose beads. The proteins modified by SUMO1 were affinity-purified from the E18 mouse brain tissue proteins and identified by HDMS. 26 potential SUM01 target proteins were identified. 20 of them are new SUMO1 target proteins; there are 74 consensus SUMO modified motif in 26 proteins (NP082280 is not in) by using the online software SUMOplot^TM Analysis Program, there is not any SUMO modified motif within only 2 proteins.Further more, to study the SUMOylation and its function of some interesting proteins, we established the technique for validating the protein SUMOylation in vivo.