Proteomics Separation And Identification Of New Technologies And New Methods

Proteomics has become a frontial discipline of analytical chemistry. Proteomics refers to the analysis of all the proteins expressed in a cell or tissue. Study of affiliation between functional proteome and genomics is of great significance. Presently application research of proteomics research focuses on differential proteome analysis of human diseases, which is designed to find key proteins with potentials to be used as markers for diagnosis or targets for meditation.Development of proteomics relies on the advances in high throughput analytical technologies including multi-dimensional separation, biological mass spectrometry and bioinformatics. Electrospay especially nano-electrospay has been the key technique in Bio-MS. Identification of all proteins in a cell or a tissue and their locations in the organism and finding out the key molecules by comparative protomics has made mass spectrometry becoming one critical tool in proteomics study.This doctorate dissertation is carried out in the course of foundation of the research lab for proteomics and establishment of experimental plateforms for the study of protein and proteomics analysis. A CE-MS/MS technique has been developed and the merits of the capillary coating have been discussed. Several homemade micro/ nanoelectrospray emitters for laboratory use were described. An online desalination for nanospray was described for the first time; An auxiliary electrode was used to improve the electric field distribution between the nESI emitter and the MS sampler so that S/N was greatly improved.This dissertation consists of four chapters and the contents are summarized as follows:In the first chapter, the key technique in the proteomics study, bio-mass and its related techniques and applications of bio-mass in the proteomics have been reviewed. Bio-mass becomes more and more important in proteomics with the development of the two soft ionization techniques namely ESI and MALDI, with the improvement of the mass analyzer, with the hyphenation of HPLC and CE, with the development of the micro-electrospay and nano-electrospay, as well as the application of ICAT. The bio-mass technique is a potential power tool for the study of the dynamic process of protein-protein reaction, the structure of genetic product, diseases related protein andfunction protein.Establishment and systematic study of an online CE separation -MS identification of proteins and peptides was presented in the chapter two. The coating of CE has also been discussed. The CZE experimental parameters were optimized with the use of a UV detector. Detection sensitivity for the standard proteins can be reached up to fettomole level via separation and identification. A sheath flow interface is constructed to hyphenate CE and ESIMS. The identification of protein is realized by MS/MS analysis of the digested peptides and then MASCOT library search. Meantime, an effective coating technique has been developed to reduce the adsorption and improve the separation efficiency. Also, some factors influencing the MS/MS results, such as protease selection, the amino acid components in the peptide and collision energy selecting and so on, have been discussed.Several homemade micro/nanoelectrospray emitters for laboratory use have been described in the third chapter. All these tips are easy to fabricate and operate. The standards have been used to test the performance of these tips. Compared with the conventional electrospray emitter, these micro/nanoelectrospray emitters have been proved to have a high sensitivity and low consumption. Stainless steal (SS) emitter (50um i.d. X 400um o.d.) was investigated as nanoelectrospray tip which enable low flow rate at 100 nl/min. The offline performance of nanospray was quite good during MS identification of standard protein digests. Therefore, SS emitter can offer a number of desirable features such as easy to fabricate, low cost as well as convenience to operate and coupling to mass spectrometer. In the meantime, MS parameters on the coexisted noncovalent complex have been discussed. Furthermore, an online technique of desalination for nanospray has been exploited for the first time. The fused silica capillary was heated and stretched to form a sharp tip (less than 1 Oum i.d.) and then 5(j.m C18 bonded porous (300A pores) was successfully packed for about 5mm long. The trypsin digested peptides were loaded onto the capillary, after desalting with water (containing 0.1% formic acid), and then flushed with 50% water, 50% acetonitrile (containing 0.1% formic acid) and made it for MS analysis. Tryptic in-gel digestion of BSA has been introduced and the method was proved to have some advantages such as easy operating and high efficiency. An auxiliary electrode has been used to modify the electric field distribution between the nESI emitter and the MS sampler. Use of the auxiliary electrode has greatly stabilized the signals and improved S/N for the nanoelectrospray.