Peptidome And Proteome Research Of Rat Choroid Plexus Tissue

The choroid plexus is the area on the ventricles of the brain where cerebrospinal fluid (CSF) is produced by modified ependymal cells. It is found in the superior part of the inferior horn of the lateral ventricles. It follows up along this boundary, continuous with the inferior of the body of the lateral ventricles. It passes into the interventricular foramen, and is present at the top of the third ventricle. The choroid plexus consists of many capillaries, separated from the ventricules by choroid epithelial cells. Liquid filters through these cells from blood to become cerebrospinal fluid. There is also much active transport of substances into, and out of, the CSF as it is made. A number of peptide and protein are synthesised directly from the CP, and CP is also responsible for transfering ascorbic acid, folic acid, vitamin B6, a small molecule peptide substances and metal ions such as oligodeoxynucleotides from blood to the CSF. In the present, transfer organization, regulation, differentiation, mechanisms of CP remain unclear. peptidome and proteome reasearch will offer a scientific materiol for discoverying of new peptide and protein with biological activities and in-depth studying functions of CP.This paper optimized the extraction methed of tissue peptide in two ways of the solvent choice for extraction and tissue pretreatment, and the this method was applied to peptidome and proteome research of rat choroid plexus tissue. We had also analyzed the proteome and peptidome expression profile of CP and the different proteome of manganese poisoning, and the relationship between protein and peptide, and found a number of key protein closely related with the damage induced by manganese in the CP.The first chapter of paper examines the CP peptidome. The first section compares tissue peptidome extraction handing with the boiling water and liquid nitrogen, and we found that more peptides were extracted with the liquid nitrogen-treated tissue, and the number of peptides found no significant change in 15min, indicating the protein degradation during processing were not emerging.Then we inspected the extraction solution. Compared with the acidic methanol solution, the number of peptides extracted by acidic aqueous solution were significantly more likely, So we have done the comparison among four kinds of acidic aqueous extraction capacity of peptides. We found the number of peptides increases as the pH decreased in acetic acid solution, saturated 2,5 - dihydroxybenzoic acid (2, 5-Dihydroxybenzoic acid, DHB), a-cyano -4 - hydroxy cinnamic acid (a-Cyano-4-hydroxycinnamic acid, a-CCA), sinapic acid (Sinapinic acid, SA). Acidic aqueous solution pH<4 can be used for extraction of CP tissue peptide, and peptide in DHB, a-CCA solution did not change within the 72h, and the peptide extraction could be saved in these solution, and SA solutin, as the pH is too high, may also lead to degradation even it could prevent corrosion.The second section is about extraction of rat choroid plexus tissue with ptimized method . we identified a total of 163 peptides in the CP of adult Sprague-Dawley (SD) rat, by using matrix-assisted laser desorption/ionization time-of-flight/ time-of- flight mass spectrometry(MALDI-TOF/TOF)and nano-liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The identified peptides were mainly derived from five precursor proteins, including ATP-synthase, cytochrome c, hemoglobin, NADH-ubiquinone oxidoreductase, beta-globin. The molecular weights of most of these precursor proteins were in the scale of 10 -20 kD. The sequences among some of the CP peptides apparently were a ladder degradation fashion with high similarity.In order to investigate the ladder degradation of peptide, analysis and classification of these peptides was done based on their chemical nature. The results showed ladder degradation of peptides compared to non-ladder degradation peptide, N terminal degradation compared to C terminal degradation, the proportion of its three properties of amino acids(acidic/basic/ hydrophobicity) remained unchanged. It indicated that whether degradated and degradated from the N or C terminal end had no relationship with their amino acid composition. There are a low proportion of acidic amino acids in terminal compared to whole peptide and a higher proportion of basic amino acids in serum peptide markers. It suggested that the nature of the terminal amino acid could reflect some specificity, may be related environment of which peptide extracted from organization or body fluids.The second chapter of this study examines the CP expressed proteome and different proteome of manganese poisoning. A total of 231 non-redundant proteins identified, mainly are binding proteins, transfer proteins with molecular skeleton, which mainly involved in cellular processes, metabolism and stress response. Cytoplasmic proteins, membrane proteins, mitochondrial proteins and cytoskeletal proteins were 45,39,38,26, accounting for 63% of the total number of identified proteins.And the binding proteins, transport proteins and the skeleton of active protein molecules were 96,33,27 respectively , accounting for 67% of the total number of identified proteins. 30% of the proteins with "high score (> 500 minutes)" were the same with the precursor proteins of peptidome, and they were mainly mitochondrial proteins. Peptides from much "high marks proteins (usually of high abundance proteins)" were not identified in the peptidome research. The level of CP protein abundance and CP peptide abundance seems no significant "correlation" relationship. The production of CP peptidome may be controled by a variety of factors, among which energy metabolism may be the major factor.By two-dimensional electrophoresis and LC-MS/MS analysis in third chapter, 448 spots were detected. By the analyses of match differentially displayed map , 40 prominent ( 30 up-regulated and 10 down-regulated) proteins were identified. Their molecular weight and isoelectric point were very similar with the prodictive values by PDQuest. In different proteome, proportions of membrane protein, mitochondria, cytoplasm, and cytoskeletal proteins were higher, and the protein ratios of binding, catalytic and transport function were also higher. These law of the GO categories was similar with the CP expression proteome pattern. These found important proteins related to catalysis, metabolism and transport has important scientific value for revealing manganese poisoning mechanism of choroid plexus tissue damage.