| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 101 === The utility of mass spectrometry (MS) for identification of post-translational modifications of proteins has boosted glycoproteomics research in recent years. However, site-specific delineation on the glycan occupancy and structure is still a challenging task due to the diversity of glycan structures, low abundance and low ionization response of glycopeptides in tryptic peptide mixtures. Nevertheless, several procedures in the shotgun-based glycoproteomic analysis platform, such as glycoprotein digestion and enrichment methods, are crucial for subsequent mass spectrometry-based glycoproteomic identification. In this study, we attempted to optimize digestion and enrichment methods to improve glycoprotein identification with six standard glycoproteins, namely horseradish peroxidase (HRP), bovine fetuin-B (Fet), chicken ovalbumin (OVA), bovine asialofetuin (FetA), bovine lactotransferrin (LF) and human transferrin (TF) and HeLa cell membrane proteins.
In the first part of the thesis, we used these six single standard glycoprotein to evaluate glycoprotein denaturing methods and pre-digestion conditions. Of the denaturing methods, four protein denaturing methods, namely, heating (95℃), alcohol(50 % 2,2,2-Trifluoroethanol, TFE), neutral material (6M urea) and salt (6M guanidine-HCl) disruption, were applied to six standard glycoproteins. Among the four methods, the use of TFE (50%) denaturation provided the most glycopeptides identified (from 1.2 to 1.4 folds) and highest glycopeptide signals (from 1.5 to 32.4 folds) in the mass spectra. Of the pre-digestion conditions tested, various concentrations of ([DTT]: [IAM]: [2nd DTT], in mM) in the ratio of 5:25:25 (condition A), 5:45:45 (condition B), 10: 25:25 (condition C), and 10:45:45 (condition D) were compared on six standard glycoproteins. The condition (A) was shown to be the most appropriate pre-digestion condition for glycopeptides identification. Hence, for the standard glycoprotein mixtures, the 50% TFE denaturing method followed by pre-digestion condition consisting of 5 mM DTT, 25 mM IAM, and 25 mM 2nd DTT was employed to further enrich the glycopeptides.
In the second part of the thesis, we aim to develop a sequential glycopeptide enrichment method by hyphened purification with non-specific HILIC (Hydrophilic interaction liquid chromatography) and sialic acid targeted TiO2 StageTips methods. On the result of six standard glycoprotein mixture, sequential enrichment by TiO2-HILIC could enrich more glycopeptides (47) compared to separate single HILIC (39 glycopeptides) and TiO2 (20 glycopeptides) enrichment method. Finally, the efficacies of the sequential enrichment approaches were evaluated using HeLa cell membrane proteins. Sequential HILIC-TiO2 (373.3 ± 37.9) and TiO2-HILIC (398.7 ± 19.5) both also efficiently enriched more glycopeptides compared to separate single HILIC (302.7 ± 41.5) and TiO2 (309.7 ± 29.2) in HeLa membrane proteins. By TiO2-HILIC, the coverage of N-glycoproteome analysis was increase 1.2 to 1.4-folds, hereby increasing the efficiency of enriching more intact glycopeptides from cell. Thus, in both standard glycoprotein mixtures and HeLa cell membrane proteins, glycoproteome identification could be expanded by our sequential glycopeptide enrichment approach.
In conclusion, thus study provided an integrated protocol with optimized denaturation and the pre-digestion conditions and sequential glycopeptides enrichment method to increase glycopeptides identification coverage while preserve intact glycan and peptide structures which were valuable information for glycoproteome research. In the future, it is expected that this rational pipeline can be utilize to benefit in-depth analysis of glycoproteome.
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