A New Strategy For Proteome Study Based On RNA Affinity And New Immobilized Enzyme

Proteomics acquire information of proteins on their composition, expression, modifications and quantification by analysis in large scale of proteins extracted from cells, tissues and organisms using mass spectrometry. Depth coverage in proteomics analysis has become an important area of proteomics research and contributes to comprehensively understanding of the complex physiological functions and pathological changes. But the limitation in detection sensitivity and scan speed of current mass spectrometry as well as the high complexity and large dynamic range of proteomic samples makes it rather difficult to identify most of the low abundant proteins which play crucial roles in the occurrence and development of many diseases. Methods commonly used for low abundant protein enrichment have limited efficiency in the broad spectrum enrichment and high throughput. Furthermore, classical separation techniques for complex protein sample only can reduce the sample complexity, but cannot increase the concentration/content of low abundant proteins. Therefore, it only results in limited coverage improvement in low abundant protein identification. Due to the above reasons, development of novel enrichment and identification strategy is one of the central issues in current proteomic researches.In living organisms, RNA binds proteins with high specificity based on their sequence and spatial structure, which plays crucial roles in cell function and gene expression. In the first part of this thesis, we have developed a new screening method based on the RNA affinity enrichment for large scale enrichment and identification of low abundant proteins. We designed and synthesized RNA probes with known sequence, then extracted RNA-protein complexes by exploiting the specific interactions between RNA and proteins, and digested RNA-proteins for LC-MS analysis, and acquired data of RNA-proteins by database search. The process of data analysis is as follows:data preprocessing, data standardization, missing data filling, data quality control, RNA-protein interactions screening, and RNA-specific binding proteins screening. We used above qualitative and quantitative data results to do data analysis for screening low abundant proteins that bind specifically with each RNA probe. With this strategy,53 RNA probes were designed and synthesized to enrich low abundant nucleus proteins from mouse liver. Based on database retrieval, the number of proteins extracted by each of the 53 RNA probes ranges from 507～268. Among these proteins,142～478 RNA-protein interactions were identified for each RNA probe and corresponding to 511 non-redundant RNA-protein interactions totally. We further analyzed all the RNA-protein interactions and finally obtained 132 low abundant proteins that having sequence dependent bind with the 53 RNA probes with high specificity. We analyzed 132 RNA-specific binding proteins with DAVID, which turned out that most of the low abundant proteins functioned well with RNA. The novel screening method based RNA affinity reagent and the strategy for low abundant proteins enrichment achieve broad spectrum and highly efficient enrichment. The novel strategy will promote current proteome identification and provides strong support for proteome analysis with deeper coverage.As the most widely used strategy in proteomic research, "shotgun" strategy uses mass spectrometry to analyze proteolytic peptides to obtain the sequence information of the corresponding proteins. Therefore, highly efficient and complete digestion of proteins is the current research hotspot and crucial step for high throughput proteomics analysis. Compared with in-solution digestion frequently used in "shot-gun" strategy, immobilized enzyme digestion has multiple advantages, such as significantly shortened digestion time, easily separated from the digestion products and recover & reuse of the enzyme. However, most immobilized enzymes currently used are prepared using solid materials as the immobilization matrix. The immobilized enzymes react with the substrate proteins in heterogeneous system. The inherent mass transfer resistance in the solid-liquid interface and steric hindrance of the solid matrix limits further improvement of the digestion efficiency.In the second part of the thesis, we have prepared a novel immobilized enzyme using soluble thermo-sensitivity polymer as the matrix material by exploiting the thermo-responsive ability of the polymer to environmental temperature changes and applied it to proteome research. The immobilized-trypsin completely dissolves in the solution and achieves homogeneous digestion at temperature above the upper critical solution temperature (UCST) with no mass transfer resistance and lowered steric hindrance, leading to increased interaction between the immobilized enzyme and protein substrates and improved digestion efficiency. While the immobilized-trypsin precipitates at temperature below UCST and can be separated from the solution and recovered easily. Therefore, the thermo-sensitive immobilized trypsin has the feature of "homogeneous digestion in high temperature and heterogeneous separation in low temperature". The digestion efficiency, completeness and stability of the newly developed immobilized trypsin was evaluated using standard protein (BSA). Sequence coverage ranged from 82% to 94% by 1 min immobilized digestion, which is higher than that of 74% obtained by 12 h in-solution digestion, which showed the immobilized trypsin obtained good effecs. Finally, the immobilized trypsin was successfully applied to fast and highly efficient digestion of complex proteome extracted from HeLa cell. The efficiency of 1 min immobilized digestion is similar to that of 12 h solution digestion. The immobilized trypsin significantly shortened digestion time and recover & reuse of the enzyme with the advantage of "homogeneous digestion in high temperature and heterogeneous separation in low temperature", which improved throughput of proteome samples and sufficiently demonstrated the application potential of this thermo-sensitive immobilized trypsin in proteomics research.Aiming at the difficulties and technical bottlenecks of proteome with limited coverage depth and low throughput, we developed new strategies based on RNA affinity and immobilized enzyme for proteomics, which provides strong support for high sensitivity and high throughput proteome research.