| With the development of proteomics research, the deep coverage of proteomes is needed in large-scale protein characterization and quantitative analysis. The deep coverage of proteomes means that more sequence of a protein and proteins of proteomes are identified by mass spectrometry identification. To achieve the deep proteome coverage and large scale quantitation, there are two main methodological strategies : one is to improve the resolution, sensitivity and speed scanning of mass spectrometers, and the other is to improve the resolution of high performance liquid chromatography.In this thesis, to improve the resolution of high performance liquid chromatography, we prepared novel reversed-phase / weak cation exchange packing materials with 3 or 1.7 .?m in diameter. The prepared mixed-mode stationary phases can overcome the disadvantage of the single-mode stationary phases, for example, analytes with similar chemical properties can be separated,and reduce the irreversible adsorption of highly hydrophobic substances be reduced. In addition, high column efficiency is achieved by reducing the particle size.This thesis consists of four parts. In the preface, we introduce the proteomics strategies, the latest development of liquid chromatography stationary phase in proteomics separation and the research field.In Chapter 2, we prepared a novel reversed phase / weak cation exchange mixed-mode chromatography stationary phase with particle size of 3 ?m. First, 3?m silica particles were modified with vinyl groups, and then the cysteine is bonded to the surface of silica microspheres by "thiol-ene" click chemistry,finally through the reaction of octadecyl isocyanate and an amino group of cysteine, a C18 chain is attached to the surface of silica particles. This kind of stationary phase has following features: C18 chain presents reversed phase chromatographic separation mechanism, and a carboxyl group on the cysteines present weak cation exchange chromatographic separation mechanism; Second,the carboxyls on cystines, carbonyl groups and amino groups are hydrophilic groups, and S atoms as the polar molecules canreduce the hydrophobicity of the functional groups, all together, the stationary phase can reduce irreversible adsorption of analytes with high hydrophobicity such as some hydrophobic peptides; Third, in the first step of the synthetic reaction of the stationary phase,vinyl groups were bonded to the reactive groups of silica microspheres, silanols on silica paticals surface can take no further reaction and sealing because vinyl trichlorosilane reagent is a kind of small molecule, the steric effect does not exist during the reaction. In addition, magnetic stirring speed, reaction reagent dosage and C18 chain containing compounds were optimized, and ultimately the optimal reaction conditions were determined. Scanning electron microscopy,infrared spectrophotometry and elemental analysis were used to characterize of the stationary phase, and the results showed that the reversed-phase / weak cation exchange mixed-mode chromatography stationary phase is complete spherical particles with uniform particle size, and carbon content up to 14.9%.In Chapter 3, the chromatographic performance of the prepared reversed phase/weak cationic exchange mixed-mode chromatographic stationary phase was evaluated. First, the separation of a mixture of benzene homologues and polycyclic aromatic hydrocarbon (PAH) proved that it has good capacity of reversed-phase separation; then by comparing with the result of ODS reversed stationary phase separation, it was proved that the hydrophobicity of stationary phase we prepared has been improved; finally, by adding a salt to mobile phase during separating standard peptide mixture, the column separation efficiency of peptides has been significantly increased, and the stationary phase was proved to have separation mechanism of weak cation exchange. Next, when the reversed phase/weak cationic exchange mixed-mode chromatographic stationary phase was used for separation of standard peptides, peptides with similar hydrophobicity but with different theoretical isoelectric points can be separated efficiently. When the prepared packing materials were packed into a 15 cm X 75?m i.d. capillary column with spraing tip by hydraulic pressure packing mode,and the capillary column was coupled with nanoLC-LTQ for a analysis, good results of separation and identification were obtained in BSA peptides. At last,the mixed-mode chromatographic stationary phase was used in the first-dimensional separation of whole Hep-G2 cell lysate, the fractions were further separated and identificated in the second dimensional capillary liquid chromatography-mass spectrometry, the results were similar to those achieved by a commercial chromatographic column.In Chapter 4, the preparation method of reversed phase/weak cationic exchange mixed-mode chromatographic stationary phase was transplanted to silicone particales with a size of 1.7 ?m. According to chromatograph velocity theory, the small particale size of a stationary phase can further increase column efficiency, therefore, a sub-2?m reversed phase/weak cationic exchange mixed-mode chromatographic stationary phase was prepared. Through characterization of scanning electron micrograph, infrared spectrophotometry and organic element analysis, the sub-2?m reversed phase/weak cationic exchange mixed-mode chromatographic packing materials with complete spherical particles, uniform particle size, carbon content up to 14.2% were prepared. Next, the assessment of chromatographic performance of the prepared sub-2-?m chromatograph stationary phase indicated that they have weak cation exchange separation mechanism while they maintain reversed separation mechanism. When the packing materials were used for separation of a standard peptide fragment mixture, high separation efficiency was achieved. In addition,because shorter columns packed with sub-2-?m chromatograph stationary phase can shorten separation time without reducing separation effect, these columns have potential application in high throughput proteomics studies. |
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