Analysis Of Serum Glycoprotemics And Subcellluar Protein Profiling After Ricin Toxin Exposure

Ricin (RT) is the most toxic natural poisons of plant proteins up to now.In these study, we are extracted from castor seeds to the gel electrophoresis of pure RT, and then we use glycoproteomics echniques and anlaysis of glycoprotein changes on Wistar rat serum glycosylation sites after RT intoxication. Additionally, we sucessfuly anlysised for the three organell protein expression profiling. These studies, in wide-depth understanding of RT in animal model and cell model based on our experimental data can be provide strong support and reference data set. Thus, this extensive comparative study also provides a data set of proteins that are present in the mitochondrion, nucleus and cytoplasm during RT-induced apoptosis.1.Purified and anlysis on Ricin toxinHomogenate by castor beans and a series of centrifugation, saturated ammonium sulfate at 80% and we can get of the crude RT. RT on the extract of the venom mixture, we used Sepharose 4B, a column for affinity chromatography to remove unwanted proteins, then we'll used Sephacryl S-200 molecular sieve column for that of RT and ricin agglutinin, after sepationed and using SDS-PAGE analysis identified the addedβ- mercaptoethanol treatment. We found that RT obtained RTA of molecular weight was 28 Kda, while RTB of obtained molecular weight was 30 Kda. Wistar rats and Hela cells were carried out LD₅₀ and IC₅₀ analysis in our experiment. The SDS-PAGE results show that we extract for electrophoresis pure of RT, Wistar rats on the 24 h LD₅₀ dose was 3.44μg/Kg, and on Hela cells 24 h IC₅₀ was 100 ng/mL.2. Shotgun glycoproteomic preliminary analysis of N-lycosylation sites in serum proteins during Ricin Toxin intoxicationWe are used 1/5×LD₅₀ of RT on a daily intraperitoneal injection of Wistar rats to monitor the behavior of mice, feed consumption, body weight changes. After a week, we are sacrified the Wistar rats and sreum collected by centrifugation, while heart, liver, spleen, lung, kidney after HE staining for paraffin sections. Serum protein concentration by BCA analysis, reduction, alkylation and adding trypsin digestion after 16 h of ultrafiltration membrane digested peptides were enriched, enriching and desalting, using the PNGase F for further enrichment of peptides after digestion, combinatione use of Shotgun analysis of serum proteins and glycosylation sites. The proteomics data were obtained and bioinformatics analysis, the protein molecular weight and hydrophilic isoelectric point and protein distribution of GO functions. The HE results of the analysis show that the Wistar rats model of ricin poisoning was successfully constructed. Using Shotgun analysis, 74 and 58 trypsin- digested proteins were identified in the control and RT-intoxication groups, respectively. Also, 33 and 50 N-glycosites and 14 and 21 glycoproteins were identified in the control and RT-intoxication groups, respectively. Mass spectrometry to identify proteins on the bioinformatics analysis revealed that trypsin group and the group glycoprotein molecular weight distribution of the protein group were 10-13 Kda and 10-80 Kda; for most of the PI distribution, and trypsin digestion glycoprotein distribution groups 5-9 and 4-11, respectively; and hydrophilicity for protein analysis showed that most of the proteins are hydrophobic proteins. Combinationed the literature analysis and our experimente result, our preliminary study only proves that the serum glycoproteins and glycosylation sites are changed. There are two glycoproteins Gc vitamin D-binding protein and Pzp alpha-1-macroglobulin may be involved in multiple organ dysfunction (MODS) after RT intoxication.3. Constrcted a Hela cell model during Ricin toxin-induced apoptosisAfter determination of the concentration of RT, MTS, trypan blue exclusion, AO/EB staining, flow cytometry, Hochst33258 staining, mitochondrial membrane potential detection, electron microscope detectioned changes in the corresponding subcellular extraction for Western blot analysis of apoptotic cells in control and RT treatment groups. The experiments results that ricin on the proliferation and growth of Hela cells significantly inhibited, IC₅₀ was 100 ng/mL, the best delivery time is 6 h; by RT before and after incubation, Hela cells with morphological characteristics biochemical characteristics of a series of changes, phosphatidylserine on the cell membrane appears valgus, the nuclei appeared shrunken and broken the case, the release of dense granular apoptotic bodies, cell nucleus ultrastructure showed swelling and occurred changes in the breakdown of mitochondrial membrane potential decline occurred before and after re-incubation, the cell membrane permeability. Western blot analysis showed that CytC release from mitochondria to cytoplasm. This results proves that we have successfully constructed Hela cell apoptosis model induced by RT exposure.4. Mitochondria, nucleus and cytoplasm of the separation and purity analysisWe are isolated the three organelles (including mitochondria, nucleus and cytoplasm) after 100 ng/mL RT-treated Hela cell apoptosis at 6 h. After collection of three organelles and frozen ultrathin sections by electron microscopy analysis. Additionally, we are used simultaneously with the nuclear histone Histone, cytoskeletal proteinβ-actin, cathepsin Cathepsin-D and heat shock protein 60 as a nucleus, cytoplasm, lysosomes and mitochondria of specific marker proteins, respectively. Using Western blot on the separation of the three organelles purity analysis. By electron microscopy, we found that the mitochondrial membrane integrity and and appear as two big middle small rod-like structure, nuclear morphology are perfected for the control group and RT treatment group; and the Western blot results show that the three organelle are prefected and not cross-contaminations for the two group.5. Comparative proteomics analysis of subcellular protein expression profiling during Ricin toxin-induced Hela cells apoptosisWe are extracted three organelles at 100 ng/mL RT treatment 6 h for the control and RT treatment groups. Using SDS-PAGE separation of mitochondria, nucleus, cytoplasm proteins and gel bands were cut and then by reduction, alkylation treatment adding trypsin 16 h, shotgun identified the mitochondria, nucleus, cytoplasm, protein expression and collected the MS data. The physical and chemical properties of the protein, metabolic protein, GO functions and hypergeometric analysis of proteins in different subcellular abundance of comparative analysis of proteins expression. Secondly, for the subcellular protein localization predictioning, we used Swiss-prot protein as the training data set in this experiment, and subcellular protein localization predictioning, according the previously paper's details. Our results show that by the 3, 107 subcellular protein mass spectrometry data (which included 297 normal mitochondrial protein, RT treatment of mitochondrial proteins 380, 826 normal nuclear proteins, 650 nuclear RT treatment proteins, 466 cytoplasm, normal protein, cytoplasmic 488 RT protein treatment group), physical and chemical properties of proteins, metabolic proteins, GO functions analysis of proteins in different subcellular expression of abundance degree of comparative analysis of protein functional annotation of the results show the normal group and RT treatment group, there are a clear difference abundance in these groups. By subcellular localization, we obtained two types of data. Firstly, we called"Location new proteins"that we are known proteins without subcellular localization annotation in the the Swiss-prot database, including a total of 1319 (45 normal mitochondria, 103 mitochondria RT treated; 271 normal nuclei, 244 nuclei RT treated; 340 normal cytoplasmic, 316 cytoplasmic RT treatment), and secondly, we called"New location"that proteins with subcellular localization annotation not in accordance with the Swiss-prot database, including a total of 30 proteins (including mitochondrial control 1; nucleus of 14 normal, 3 the nucleus RT treatment; cytoplasmic normal 6, 6cytoplasmic RT treatment group).6. Summary and OutlookIn this chapter, we are summarized in detail the findings of this experiment, combined with the latest literature mining and analysis of the current research progress. Therefore, a reasonale analysis the inadequacies, make efforts to improve the experiment direction. Additionally, we are have been a new ideas for my post-doctoral of further research career comes from a accumulation of theoretical knowledge fundmental.