The Development Of RNA Based Affinity Reagents For In Depth Proteomic Study

With the accomplishment of human genome sequencing project, the research oflife science enters the post-genome era. As the hotspot area in post-genome era,proteomic research has made a rapid development. Facilitated by the research on thefunction, interaction and dynamic changes of proteins in the overall protein levels, themechanism of physiological activities and the nature of pathological changes can bebetter revealed. Biological mass spectrometry, widely used in proteomics research,greatly promotes the development of large-scale identification of proteome. However,the high complexity, extremely large dynamic range of proteomic samples and thelimitation of the detection sensitivity makes the identification of low abundanceproteins particularly difficult. Especially those having key funcations in cellular signaltransduction regulation and clinical diagnosis are often overwhelmed by the high andmedium abundant proteins. Therefore, the development o f novel technologies forlow-abundance proteins enrichment and identification in large scale is one of theurgent demands in current proteomic research.In order to break the technological bottlenecks in current strategy for large scalelow abundance protein enrichment, we have exploited the characteristic affinity ofRNA towards proteins and developed RNA-based affinity reagents for the enrichmentof low-abundance proteins. This strategy is expected to provide technical support forthe human proteome profiling with deeper coverage. In the first part of this thesis, weestablished the endogenous mRNA-based strategy for large-scale low-abundanceproteins enrichment. This enrichment approach was combined with the high pHreverse phrase chromatograph separation and successfully applied to the deepcoverage of mouse liver proteins, which resulted in more than4000identified proteinsand1352new discovered mouse liver proteins compared with the PaxDb database.The proportion of low-abundance proteins identified is significantly higher than thatobtained without enrichment, especially in the extremely low abundance range of lessthan20ppm. Among identified proteins, there are429RNA binding proteins, whichcovers53.4%of the known mouse RNA binding proteins and exceeds the literaturereported coverage percentage. After comprehensively analyzing the identified proteins,information regarding the protein domain, function and location were obtained, which provides new understandings for the RNA binding proteins.In view of the successful application of the endogenous mRNA-based proteinenrichment strategy, we have developed the exogenous RNA aptamer-based lowabundant target protein enrichment strategy. In the second part of this study, efficientenrichment of low-abundance protein was achieved and a database containing theinformation of the enriched proteins and corresponding RNA aptamer was constructed.By optimizing the enrichment strategy and using13aptamer sequences,low-abundance proteins in mouse liver were successfully enriched. After integratingall the data, we identified595low-abundance proteins with the sequence informationof the corresponding interacting RNA. The combined application of the endogenousand exogenous RNA-based protein enrichment strategy can effectively improvecurrent proteome identification coverage and provide strong technical support for theimplementation of the Human Proteome Project next.