質譜分析有效應用於醣質定序與硫酸化鑑定

Addressing the technical limitation in glycomic sequencing by mass spectrometry

于心宜(Shin-Yi Yu)

指導教授 ： 邱繼輝

國立臺灣大學/生命科學院/生化科學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01714

摘要

黏蛋白種類 (mucin-type) 的醣化現象 (O-glycosylation)，其醣類結構在眾多修飾蛋白質的醣類中最為複雜，因此在醣質體結構圖譜 (glycomic mapping) 及定序的領域中仍屬於複雜和難以解決的一環。O-glycans 的結構組成複雜的原因在於由不同種類的核心 (core) 結構、延伸的醣鏈骨架及末端結構有眾多不同的醣質及化學修飾 (岩藻醣 fucose、唾液酸 sialic acids、硫酸化 sulfation)。現今仍未有快速及有效的O-glycans 結構分析，因此希望藉著質譜儀的分析可以快速得到細胞內O-glycans醣質現象的輪廓，且藉著串聯式質譜快速地得到O-glycans 的核心及末端結構的修飾，其為與外界作用重要的因子。所以先用已廣泛被研究的牛頜下腺黏蛋白 (bovine submaxillary mucins) 當作標準品得O-glycans後，(1) 將醣經甲基化處理後，用串聯式質譜儀建立特殊斷片模式以快速分析O-glycans結構，(2) 藉由碘酸鈉 (periodate oxidation) 的化學處理，連結質譜儀分析得O-glycans，其 3 arm 和 6 arm 的醣類組成，(3) 用陰離子交換樹酯建立純化方法 (fractionation) 以偵測微量和帶負離子的醣。接著取罹患肺氣囊腫的病人唾液和來自於健康病人的呼吸道組織切片細胞培養的分泌物用已建立的純化方法分離出帶有硫酸根的醣，同樣的用串聯式質譜建立未經過處理和經過甲基化處理帶有硫酸根的O-glycans的特殊斷片模式。結合樣品前處理和質譜儀分析，我們可以進一步快速偵測不同樣品來源下，帶有硫酸根的醣其差異性表現。

接著，將已建立好快速分析O-glycans結構的方法應用在富含O-glycans的鼠舌下腺醣蛋白 (rat sublingual glycoproteins) 和人類子宮囊醣蛋白350 (human ovarian cyst glycoprotein350)，檢視是否能印證利用凝集素 (lectin) 得知的醣纇結構。利用質譜分析得知鼠舌下腺醣蛋白的O-glycan結構特徵為core 3 或 core 4 為主的核心結構，延伸出去的是type 2 LacNAc醣鏈骨架且其末端有唾液酸修飾、無硫酸根修飾。相反的，人類子宮囊醣蛋白350 的 O-glycan結構主要以core 1 或 core 2 為主的核心結構，延伸出的醣鏈為延展性 (extended) type 1 LacNAc醣鏈並從半乳醣殘基 (galactose residue) 6 arm分支出 type 2 LacNAc醣鏈，其末端半乳醣醣基有唾液酸的修飾。現今利用質譜儀的定序分析可以支持前人所預測人類子宮囊醣蛋白上的O-glycan結構；且利用已建立的特殊斷片模式只偵測到Lewisa/x 的斷片可以得知樣品來源是來自non-secretor 的提供者。此外，在負離子模式下的質譜儀分析發現人類子宮囊醣蛋白350 有硫酸根修飾的 O-glycans 存在。除了成功鑑定數個黏蛋白上的未知醣類結構外，主要想闡示可藉由質譜儀及串聯式質譜分析快速的分析醣質體內醣化現象的輪廓及其主要核心結構和末端結構修飾；配合凝集素 (lectin) 的研究可以進一步有意義的探討影響醣化作用的醣轉移酵素脢的作用機制。

關鍵字

質譜分析 醣質定序

並列摘要

Among the diverse range of protein glycosylation, mucin-type O-glycosylation remains one of the most refractory to structural mapping and sequencing due to its extreme heterogeneity in core types, extended backbone chains and terminal epitope modifications introduced by various specific combinations of fucosylation, sialylation, and sulfation. Current analytical limitations are well appreciated and further development of enabling techniques are needed to facilitate comprehensive glycomic profiling by advanced mass spectrometry (MS). To this end, we first use the commercially available bovine submaxillary mucins to derive a wide range of O-glycans as standards to establish specific fragmentation patterns in collision induced dissociation (CID) MS/MS analysis of permethyl derivatives, before and after periodate oxidation, and to set up sample fractionation scheme to detect low abundant and negatively charged species. A first screen strategy by MS was successfully developed which led to identification and sequencing of several novel structures not previously reported. Sulfated O-glycan fractions from the sputum of cystic fibrosis patients and total mucin secretions of normal human tracheo-bronchial epithelial cell culture were then tackled to optimize for sample pre-treatment procedures, chemical derivatization, and to define MS and MS/MS patterns specific to sulfated glycans. This work demonstrated that strategic glycomic mapping is feasible for sulfated glycans in which differential expression pattern can be readily established.

Finally, the methodology developed was applied to define the characteristic features of O-glycans from rat sublingual glycoprotein (RSL) and human ovarian cyst glycoprotein 350 (HOC350) in the context of providing the structural basis for their observed lectin binding properties. The O-glycans of RSL mucins were found to contain exclusively core 3 and 4 types extended with sialylated type 2 backbone chain, but not sulfated. In contrast, the O-glycans of HOC350 are based on core 1 and 2 types, with branched extended type 1 backbone chain substituted by mostly sialylated type 2 units at the 6 positions of the internal Gal and reducing end GalNAc. The previously proposed composite internal structures for HOC is thus fully supported by current sequencing data while the fucosylated and/or sialylated terminal epitopes identified is consistent with the non secretor status of the donor. Only Lea/x could be detected and not the ABO(H) or Ley/b determinants. In addition, sulfated O-glycans were also identified through separate modes of MS analysis in conjunction with chemical derivatization and sample enrichment scheme developed. This is the first ever report on the presence of sulfated O-glycans in human ovarian cyst fluids, as well as the first definitive mapping of sialylated termini. With the glycomic map in hand, existing and future functional and lectin binding studies can now be meaningfully dissected at the molecular level.

並列關鍵字

mass spectrometry MALDI-TOF O-glycans

參考文獻

Bierhuizen, M. F., Mattei, M. G., and Fukuda, M. (1993). Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1,6-N-acetylglucosaminyltransferase gene family. Genes Dev 7, 468-478.

Bird, G. W. (1990). Lansteiner Award. Some aspects of serological specificity. Transfusion 30, 390-400.

Breg, J., Van Halbeek, H., Vliegenthart, J. F., Klein, A., Lamblin, G., and Roussel, P. (1988). Primary structure of neutral oligosaccharides derived from respiratory-mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500-MHz 1H-NMR spectroscopy and quantitative sugar analysis. 2. Structure of 19 oligosaccharides having the GlcNAc beta(1----3)GalNAc-ol core (type 3) or the GlcNAc beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol core (type 4). Eur J Biochem 171, 643-654.

Brockhausen, I. (1999). Pathways of O-glycan biosynthesis in cancer cells. Biochim Biophys Acta 1473, 67-95.

Brockhausen, I. (2003). Sulphotransferases acting on mucin-type oligosaccharides. Biochem Soc Trans 31, 318-325.