Technology And Method Development For Sample Pretreatment And Automated Analysis In Proteome Research

Although proteomic technologies have made major progress in the last decades, it still is a big challenge to develop more efficient methods and technologies in proteome research, including the development of the methods and technologies for automated analysis system and efficient extraction of proteins in different samples.An approach was developed to automate sample introduction forμLC-MS/MS analysis using strong cation exchange (SCX) trap column. It was observed that higher performance of separation could be achieved with the system using SCX trap column than the conventional system using C18 trap column.Utilizing strong anion exchange (SAX) trap column, a fully automatedμLC-MS/MS system was introduced to analyze the enriched phosphopeptide samples. The performance of this system was evaluated by analyzing the tryptic digest of mouse liver lysate enriched by ZrO₂ nanoparticles. Compared with C18 trap column system, it was found that higher performance could be achieved with the SAX trap column system.An LMA-EDMA monolithic column was applied as the trap column and analytical column to set up the automated system for proteome analysis. Its performance was evaluated by analyzing the tryptic digest of BSA and yeast lysate. The automated system with monolithic column allowed sample to beloaded in short time and, in the mean time, kept high separation performance.A protein extraction method using 6 M guanidine hydrochloride with heating was developed for proteome analysis of formalin-fixed tissue. The peptides and proteins identified from formalin-fixed tissue were first comprehensively compared with those identified from frozen-fresh tissue. It was found that majority of peptides identified from fixed tissue were unmodified and proteome coverage by shotgun analysis of the fixed tissue was not obviously compromised by the formalin fixation process.A sequential protein extraction method was developed for large scale proteome analysis of bone tissue. Automated 2D-LC-MS/MS analysis of the tryptic digests of collected protein fractions resulted in the identification of 6202 unique peptides matched 2479 unique proteins. The identified proteins revealed a broad diversity in the protein identity and function, which was also the first study for large-scale proteomic analysis of bone, might be helpful for clarifying the mechanisms of bone diseases.