Separation And Characterization Of The Yeast Protein

The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome.To further understand the roles of protein glycosylation in eukaryotes,glycancontaining proteins in yeast were identified.The strategies for glycosylation analysis by mass spectrometry(MS)have become a basic and effective tool for analyzing glycoproteins.The detection of glycopeptides by MS is often hindered by the coexistence of the non-glycopeptides.Therefore,it is necessary to selectively enrich glycopeptides from peptide mixture prior to MS analysis.To this end,a customized hydrophilic matrix named 'XAmide' in our group to improve the effectiveness of HILIC-based methods was introduced.In this dissertation,the separation of ovalbumin glycoprotein on the homemade C8MHA column was investigated to demonstrate the separation of a glycosylated protein.Moreover,use of the C8MHA column results in good reproducibility in glycolprotein separation without changing separation efficiency and resolution,at the same time column was used for separation of inulinase glycoprotein.Furthermore,the selectivity of XAmide in glycopeptides enrichment was evaluated from two aspects:selectivity and gycosylation microheterogeneity coverage.13 glycopeptides in RNase B digest and 16 glycopeptides in IgG digest have been identified by using XAmide.In addition,151 glycopeptides of AGP were found in the fraction which is enriched by XAmide.To study the yeast glycosylation information,inulinase glycoprotein was purified by the C8MHA column from a yeast fermentation broth and analysed.The glycoproteomics method was used to study the N-linked glycopeptides in the inulinase digest.Overall,our studies greatly extend the understanding of protein glycosylation in eukaryotes through the identifying of glycoproteins in yeast,and it is believed that C8MHA column and XAmide will become useful tools in glycoproteomic studies.