Establishment And Application Of Technological Platform Of Multi-Dimensional Liquid Chromatography-Mass Spectrometry In The Study On Proteome

Proteomic technological platform of high throughput, high sensitivity and automation is always very import and difficult to be built. The research, one of many hot spots in the area, not only challenges us to solve the problems but also provide us a good chance to conduct an investigation in the methodology. As the unit of chairman of international human liver proteome project, we want to lead all scientists in the field to tackle key problems and do our best to contribute to project, and are simultaneously responsible for probing method of curing liver disease and the task of prospering our country and making nationality stronger. So technical innovation should be made to overcome the drawback the methods recently employed have. The ultimate goal is to establish a reliable and practical method to meet the requirements of proteomic study. Therefore, we have done research into the establishment of multidimensional liquid chromatography coupled with tandem mass spectrometry.Capillary columns are key parts in the high efficient separation of complex protein mixture. But the price of commercially available capillary columns is very expensive and there are many problems in the application of capillary columns. So they can not meet the demands of high throughput of proteome research. A method developed for frit-making and packing of capillary chromatographic columns was a simple, quick, high-performance and low cost technology. It was easy to operate and an expertise. All capillary columns in different specification can be packed with different kinds of packing materials with the method. These capillary columns prepared with the newly developed technique can be employed to perform the separation of small molecule, biopolymer or peptide mixture and so on. Also these capillary columns made with the method can be used in various capillary electrochromatography or capillary electrophoresis. This technique is patented and patent number is 03121042.2.High-performance separation of peptide mixture ploys an important role in the identification of proteins and in the study of their interactions in the field of proteomics. But there are not many reports on the separation of complex peptidemixture. Therefore, the relationship between peak capacities or sensitivity and column lengths for the separation of peptide mixture on two reversed-phase columns with different lengths by high-performance reversed-phase liquid chromatography was studied and the effect of gradient times on peak capacity and peak widths was examined too. The experimental results showed that column lengths have significant effect on peak capacity and detection sensitivity. Furthermore, longer gradient times increase not only the peak number, but also peak width, which is very favorable for the detection of much more peptides when separating and identifying them by capillary liquid chromatography combined with tandem mass spectrometry.Severe acute respiratory syndrome (SARS) formidably threatens the safety of the people all over our country and societal stability. To probe into disease mechanism coming on and to develop diagnosis reagent, preventative bacterin and therapy drugs, various structural proteins including nucleocaspid protein, spike protein and membrane glycoprotein of SARS-CoV in the culture supernatant of virus infected-Vero E6 were identified by two dimensional (SCX and RPLC) capillary liquid chromatography (2D-CapLC) coupled with ESI-QTOF MS/MS and analytical reversed-phase high-performance liquid chromatography-hybrid CapLC with ESI-QTOF MS/MS. MALDI-MS determination showed that relative molecular weight of intact N protein is 45929 Da, which is very much close to its theoretically calculated molecular weight (45935.38 Da ) based on the amino acid sequence with the first amino acid methionine in N-terminus depleted and second serine acetylated, indicating that phosphorylation and glycosylation do not happen or the amount of phosphorylated and glycosylated protein is very low in the predicted phosphorylation and glycosylation sites within infected cells. Further study showed that possible degradation of N protein occurred and the mechanism will be investigated. In addition, a new technological method was developed for the separation and identification of proteome of a simpler biological sample which is described as follows: protein mixture was first separated; then the fractions were concentrated and proteolysed, and finally peptide mixture was further separated and identified by capillary reversed-phase chromatography-tandem mass spectrometry. The cover rate of proteinidentification was enhanced greatly.The complexity of human liver is merely less than that of brain which is the largest of all organs in human body. Human liver plays an important role in the life movement in the body. Furthermore, human fetal liver in the age of 4~6 months is the major source of hematogenic cell and immunologically ancestral stem cell, so the study on the proteins of fetal liver is significant in academic research and in practice. Double-gradient mode was first presented and employed in the separation of intact protein m ixture. N ew m ethod i ncluding i ntact p rotein s eparation b y t wo-dimension chromatography, one-dimension capillary reversed-phase chromatography and tandem mass spectrometry w as developed. T he tryptic digests of 80 fractions from three p H ranges were s eparated and i dentified b y using t he m ethod. 4 373 p eptides were identified and assigned to 659 proteins among which the most are mitochondrial proteins. The number was first r eported in the world and the research work lays a solid basis for the study on these protein functions and biological significance.