Application Of Multidimensional Chromatography And Mass Spectrometry In Proteomic Research

A resurgence of interest in the human plasma proteome has occured in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. Proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed phase liquid chromatography coupled with linear ion trap mass spectrometry. With meeting stringent criteria, a total of 1292 distinct proteins were successfully identified. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. We coupled immobilized metal affinity chromatography with the linear ion trap mass spectrometry to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although the binding of nonphosphorylated peptides to the IMAC column is obvious, the improvement in resolution, high-speed scan mode and good MS/MS of linear ion trap have been contributed to the phosphoprotein identification. Further analysis demonstrates the MS/MS/MS is necessary to exclude the false-match of the MS/MS. The use of linear ion trap enables nanoflow HPLC MS/MS and MS/MS/MS to have a good potential in phosphoproteome research of relatively complicated samples.Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. Strong anion exchange chromatography is an alternative to this aim, immobilized metal-ion affinity chromatography has widely applied in purification of phosphopeptides , but many phosphopeptides have poor even no retention on the poly styrene based material in desalting process of IMAC. A novel integrated tandem RP-SAX system was designed for phosphoproteomics. After peptides loaded on the integrated column under alkaline conditions, and eluted with a series of citric acid buffers with different pH, 37 phosphpeptides from mouseliver, corresponding to 46 phosphorylation sites were identified successfully in the eluted fractions under acidic buffers by capillary LC-ESI MS/MS, indicating polystyrene-based stationary phase of great potential in phosphoproteomics research.Preparation of a poly (styrene-co-divinylbenzene-co-methacrylic acid) monolithic stationary phase for the use in CEC has been improved by optimizing the polymerization conditions. It is observed that the reaction time strongly affects column efficiency, while the proportion of isooctane in porogen influences peak symmetry of some solutes seriously. The lifetime of the monolithic columns prepared mainly depends on the pH of buffers used. Reproducibility of EOF from batch to batch columns are lower than 2.8% (RSD). Unlike other types of capillary electrochromatographic monoliths, a pH-dependent EOF was observed on this type of column. Separation of various types of compounds including aromatic hydrocarbons, hormones, anilines, basic pharmaceuticals, and peptides were achieved. The facile preparation and wide application of this monolithic column may make styrene-based polymer a potential stationary phase in CEC.