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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 邱繼輝(Kay-Hooi Khoo) | |
| dc.contributor.author | Chih-Hsuan Yeh | en |
| dc.contributor.author | 葉芷瑄 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:50:24Z | - |
| dc.date.available | 2025-07-31 | |
| dc.date.copyright | 2020-08-14 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-07-31 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55184 | - |
| dc.description.abstract | 蛋白質醣基化是一種常見的轉譯後修飾，在特殊位置的O型醣基化修飾調控了多種蛋白酶解的功能，例如前蛋白的活化和細胞膜表面的胞外蛋白釋放，但與N型醣基化相比，利用質譜儀分析O醣基化在特定絲胺酸(Serine)和息寧胺酸(Threonine)上有較大的難度，像是當黏液素蛋白(mucin)被胰蛋白酶(trypsin)切割後，胜肽鏈上有可能仍帶有多個絲胺酸和息寧胺酸，但因O型醣鏈加上的位置沒有特殊的胺基酸序列，所以無法光藉由胺基酸序列得知正確位置。此外，目前尚沒有可以自胜肽鏈分離O型乙酰半乳糖胺類(O-GalNAc)的酵素，以及質譜分析中常用的碰撞誘導解離(CID)斷裂方式易使整個醣鏈掉落且不留下痕跡也都是使質譜分析中無法辨別特定位置的原因。在艾克曼嗜黏蛋白菌(A. Muciniphila)中發現的一種新的O型蛋白酶(或稱OpeRATOR)有機會成為識別O型醣基化位置分析中的有力工具。在近期的報導中，OpeRATOR被發現可以切在胜肽鏈上帶有O型醣基化的絲胺酸和息寧胺酸之N端，當胜肽鏈固定在平台上時，利用OpeRATOR作用後，可以特定產生只有一個絲胺酸或息寧胺酸在N端的醣胜肽，使後續的質譜分析相對容易。
本研究中，使用微量液相層析串聯質譜儀利用更高能量碰撞誘導解離(HCD)以及電子傳遞更高能量碰撞誘導解離(EThcD)兩種斷裂模式以提供可靠的斷片資訊，並首先分析OpeRATOR在液體中作用於常用的標準醣蛋白：小牛胎球蛋白(bovine fetuin) 來研究OpeRATOR的特異性。研究中發現OpeRATOR不僅會切在帶有唾液酸或沒有醣基化的絲胺酸和息寧胺酸之N端的不絕對特性之外，也會有沒切除到帶有O型醣鏈的絲胺酸和息寧胺酸之失誤。酰肼衍生化(acetohydrazide derivatization)和串聯質量標籤標記(TMT labeling)對帶有唾液酸的醣胜肽鏈和對電子傳遞更高能量碰撞誘導解離斷裂的影響也有更進一步的瞭解。接著，將OpeRATOR使用在黏液素蛋白家族中的粘蛋白-16(MUC-16)和足糖萼蛋白(Podocalyxin)上，並與先前研究發表的另一種黏液素特定O型蛋白酶(或稱StcE)做比較，由於這兩種蛋白酶的切位特異性及對醣基化的要求不同，導致可找到的醣位點不完全相同。另外，也將它們所找到的醣位點與經基因工程改造的SimpleCell所找到的醣位點作比較。最後，在有或無ST8Sia6(一種負責做出雙唾液酸(diSia)的糖轉移酶)一同轉染所產生出的醣蛋白(CD79a, FcγRIIb, FcμR和CD20)中，利用胰蛋白酶加上OpeRATOR與其他蛋白酶的組合比較下，在分析O-醣基化有最好的表現，並找到帶有雙唾液酸的特定位點，且發現在ST8Sia6轉染的醣蛋白中雙唾液酸都有增加的趨勢。 總結來說，OpeRATOR 在O 型醣基化分析中是個很好用的工具，而在液體中作用相比於固定在平台上可以減少樣品的遺失且提供更完整的資訊。在OpeRATOR 的作用下，不只電子傳遞更高能量碰撞誘導解離斷裂出可以判斷位置的斷片，更高能量碰撞誘導解離也有機會用來判定O 型位點。利用微量液相層析串聯質譜儀分析OpeRATOR 作用下的醣蛋白，判斷O 型位點將變得更加容易且有效，並可以進一步大規模分析蛋白質體中的O 型醣基化。 | zh_TW |
| dc.description.abstract | Glycosylation is one of the most abundant posttranslational modification of proteins. Site-specific O-glycosylation regulates several proteolytic processing such as proprotein activation and ectodomain shedding. Compared to N-linked glycosylation, identification of site-specific protein O-GalNAc glycosylation at Ser/Thr by mass spectrometry analysis is significantly more challenging. In particular, it has no distinctive sequon for a priori localization onto one or more of the many possible Ser/Thr sites typically carried on tryptic peptides derived from mucin domain. This is compounded by the lack of a pan-specific O-GalNAc glycan-releasing enzyme and facile loss of the entire O-glycans via common modes of CID fragmentation without incurring a scar, which defies MS/MS sequencing and site determination. A newfound endo-O-protease from Akkermansia muciniphila, commercially branded as OpeRATOR, is a potentially very powerful tool that would enable site-specific O-glycosylation mapping. OpeRATOR was recently reported to recognize O-GalNAc glycan and cleave its peptide carrier at the N-terminus of a glycosylated Ser/Thr conjugated on solid-phase platform. A complete digestion would in principle enrich and produce glycopeptides with O-glycosylated Ser/Thr located only at the N-termini and not elsewhere, thus making subsequent MS/MS analysis relatively easy.
In this study, nanoLC-MS/MS using both HCD and EThcD MS2 fragmentation modes provided the evidence in support of the deduced specificity, the OpeRATOR in-solution digestion against the gold standard bovine fetuin was benchmarked investigated. The non-complete digestion of cleaving non-glycosylated and sialylated site, and missed cleavage of glycosylated Ser/Thr were evaluated. The effect of acetohydrazide derivatization and TMT0 labeling on sialylated glycopeptides and EThcD fragmentation were further investigated as well. The application of OpeRATOR on mucin-type glycoproteins, MUC16 and Podocalyxin, were compared with previous work employing another mucin-specific O-protease, StcE. These two O-proteases have different specificity preference and glycan requirements that the identified O-glycosites were not highly overlapping. Moreover, the glycosites identified by OpeRATOR and StcE on MUC16 and Podocalyxin were further compared with those identified by use of genetic engineered SimpleCell strategy. Finally, in applications to analyses of recombinant glycoproteins, CD79a, FcγRIIb, FcμR and CD20, with or without co-expression of a terminal di-sialyl unit forming sialyltransferase, ST8Sia6, the use of trypsin plus OpeRATOR was shown to enable the best performance of identifying the target disialylated O-glycopeptides compared to the use of other protease combination. Taken together, OpeRATOR is a useful tool for site-specific O-glycosylation analysis to provide more comprehensive information. Not only EThcD can generate the fragment ions required for site-specific O-glycosylation, HCD can also localize the glycosites when used in combination with OpeRATOR digestion. Through LC-MS/MS analysis of OpeRATOR digested glycoproteins, site-specific O-glycosylation can be identified easier and more efficiently, showing great potentials for further applications to large-scale O-glycoproteomics. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T03:50:24Z (GMT). No. of bitstreams: 1 U0001-3007202022592000.pdf: 7901153 bytes, checksum: c9c2bb4c2d08730861595f0ee6501304 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 摘要 …………………………………………………………………………………………………………………………………………………………………………………………………………I ABSTRACT ………………………………………………………………………………………………………………………………………………………………………………………III Table of Contents ……………………………………………………………………………………………………………………………………………………………………V List of Figures ……………………………………………………………………………………………………………………………………………………………………VII List of Tables …………………………………………………………………………………………………………………………………………………………………………IX Abbreviation …………………………………………………………………………………………………………………………………………………………………………………X Chapter 1 – Introduction …………………………………………………………………………………………………………………………………………………1 1.1 Proteins glycosylation ……………………………………………………………………………………………………………………………………………1 1.2 Analytical strategy of glycoproteomics …………………………………………………………………………………………………3 1.2.1 Mass spectrometry-based glycoproteomics …………………………………………………………………………………………3 1.2.2 Difficulties of O-glycosylation analysis ………………………………………………………………………………………5 1.2.3 Importance of site-specific O-glycosylation analysis ………………………………………………………7 1.3 Current strategies and tools for site-specific O-glycosylation analysis …………8 1.3.1 EThcD ……………………………………………………………………………………………………………………………………………………………………………………8 1.3.2 SimpleCell ……………………………………………………………………………………………………………………………………………………………………10 1.3.3 StcE ……………………………………………………………………………………………………………………………………………………………………………………12 1.3.4 OpeRATOR …………………………………………………………………………………………………………………………………………………………………………14 1.4 Specific aims …………………………………………………………………………………………………………………………………………………………………18 Chapter 2 – Materials and Methods ………………………………………………………………………………………………………………………19 2.1 Materials ……………………………………………………………………………………………………………………………………………………………………………19 2.2 In-solution and in-gel proteolytic digestions ……………………………………………………………………………19 2.3 Acetohydrazide derivatization ………………………………………………………………………………………………………………………19 2.4 TMT0 labeling …………………………………………………………………………………………………………………………………………………………………20 2.5 Glycopeptides analysis by LC-MS/MS …………………………………………………………………………………………………………20 2.6 Mass spectrometry data analysis …………………………………………………………………………………………………………………21 Chapter 3 – Results ……………………………………………………………………………………………………………………………………………………………22 3.1 Identification of the characteristics of OpeRATOR …………………………………………………………………22 3.1.1 Characterization of OpeRATOR digested bovine fetuin glycopeptides …………………22 3.1.2 O-glycosylation pattern and LC-MS/MS elution time ……………………………………………………………32 3.1.3 Impact of sialic acid acetohydrazide derivatization ………………………………………………………34 3.1.4 Impact of TMT0 labeling …………………………………………………………………………………………………………………………………38 3.2 Applications of OpeRATOR on mucin-type glycoproteins …………………………………………………………46 3.2.1 MUC16 and Podocalyxin digested by OpeRATOR ………………………………………………………………………………46 3.2.2 Non-specific cleavage by OpeRATOR ………………………………………………………………………………………………………49 3.2.3 Comparing the use of OpeRATOR to other O-glycosylation studies …………………………51 3.3 Applications of OpeRATOR on mouse B-cell disialylated-glycoproteins …………………60 3.3.1 OpeRATOR allowed better O-glycosylation analysis than other proteases ………60 3.3.2 DiSia on the recombinant CD79a, FcγRIIb, and FcμR ……………………………………………………………62 Chapter 4 – Summary and Discussion ……………………………………………………………………………………………………………………76 References ……………………………………………………………………………………………………………………………………………………………………………………79 | |
| dc.language.iso | en | |
| dc.subject | 醣胜肽 | zh_TW |
| dc.subject | 質譜儀分析 | zh_TW |
| dc.subject | 電子傳遞更高能量碰撞誘導解離斷裂 | zh_TW |
| dc.subject | O 型蛋白酶 | zh_TW |
| dc.subject | 特定位置O 型乙酰半乳糖胺醣基化判斷 | zh_TW |
| dc.subject | Site-specific O-GalNAc glycosylation | en |
| dc.subject | O-protease | en |
| dc.subject | Glycopeptide | en |
| dc.subject | EThcD fragmentation | en |
| dc.subject | Mass spectrometry analysis | en |
| dc.title | 利用O型蛋白酶促進質譜分析辨別特異位點之O型醣基化 | zh_TW |
| dc.title | Identification of site-specific O-GalNAc glycosylation by ways of an O-protease-facilitated mass spectrometry analysis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳玉如(Yu-Ju Chen),徐尚德(Shang-Te Danny Hsu) | |
| dc.subject.keyword | 特定位置O 型乙酰半乳糖胺醣基化判斷,O 型蛋白酶,電子傳遞更高能量碰撞誘導解離斷裂,醣胜肽,質譜儀分析, | zh_TW |
| dc.subject.keyword | Site-specific O-GalNAc glycosylation,O-protease,EThcD fragmentation,Glycopeptide,Mass spectrometry analysis, | en |
| dc.relation.page | 85 | |
| dc.identifier.doi | 10.6342/NTU202002133 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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