電灑法二維離子阱質譜於醣結構及醣蛋白醣化之研究

Structure Characterization of Oligosaccharide and Protein Glycosylation Analysis by Electrospray Linear Ion Trap Mass Spectrometry

白珮瑾(Pei-Jing Pai)

指導教授 ： 何國榮

國立臺灣大學/理學院/化學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01072

摘要

醣化為蛋白質最主要的轉譯後修飾之一，醣蛋白上的醣鏈在蛋白質的結構與功能上，扮演了重要的角色。本研究利用近年所開發之新一代離子阱-二維離子阱質譜儀進行醣蛋白醣結構以及醣化之分析，首先於醣鏈結構分析上，醣蛋白以醣胜肽酶F進行切醣，沿用實驗室所開發之ABEE（p-aminobenzoic acid ethyl ester）閉環標記-負離子電灑法多次質譜/質譜分析寡醣鍵結點之方法，以不經過層析分離的方式直接分析醣蛋白之醣鏈結構，此方法可解出核糖核酸酶BMan5和運鐵蛋白醣鏈A2之完整結構，以及核糖核酸酶B除了1-3分支上雙糖外Man6之結構，並應用於核糖核酸酶BMan7之同質異構醣鏈比例之判定。
醣蛋白之醣化可分為兩個重點：第一在於觀察個別醣化位置上醣鏈之分佈，第二為醣化位置之判定。本研究以醣胜肽為分析的對象，保留個別醣化位置的資訊，以胰蛋白酶（trypsin）水解醣蛋白，利用纖維素微晶體（cellulose microcrystalline）純化醣胜肽，以增加醣胜肽於樣品中之相對量，減少醣胜肽的訊號受到共溶析（coelute）胜肽之抑制，再以毛細液相層析（CapLC）結合電灑法（ESI）二維離子阱質譜儀分析純化後的醣胜肽，來得知各別醣化位置上醣鏈的分佈。醣化位置之判定是利用二維離子阱多次質譜/質譜及掃描速度快之特性，得到醣胜肽上來自胜肽的特徵裂片離子，經由資料庫搜尋比對即可達到蛋白質之鑑定並得知醣化的位置。以核糖核酸酶B來探討醣胜肽於多次質譜/質譜下之斷裂行為，從中歸納出自動化多次質譜/質譜判定醣化位置之方法，並應用於分析運鐵蛋白之醣化，可成功判定兩個醣化位置，而由逆相毛細管液相層析結果發現於同一個醣化位置之醣胜肽，其滯留時間會依據唾液酸的個數來排列，相同唾液酸個數之醣胜肽會出現於同一個層析峰中，因此最後利用選擇離子層析峰之強度，可以得到個別醣化位置上醣鏈分佈之資訊。將此方法應用於醣化更為複雜之α-1酸性醣蛋白（來源物種：牛）之醣化分析，綜合去唾液酸以及保留唾液酸之醣化分析結果，可得到個別醣化位置醣鏈之分佈，且利用二維離子阱質譜儀具有多次質譜/質譜的特性，達到醣化位置之判定。
毛細液相層析相當耗時且設備昂貴，並且容易有樣品殘留的問題，因此開發一短填充探針（填充1.5 cm C18顆粒）進行快速分離，以縮短分析時間，並嘗試不使用HPLC之液相幫浦，來降低整體設備所需之費用，且此短填充探針的成本相當低，亦可作為拋棄式來克服樣品殘留的問題。然而，於醣化位置判定上，由於短填充探針所分離之層析峰相當窄，因此難以進行多次質譜/質譜的方式來判定醣化位置，利用氮連接型式醣蛋白醣鏈之基本結構並搭配胜肽序列上之規則，而設計出僅以二次質譜/質譜之結果即可判斷醣化位置之方法。以此方法可成功的判定核糖核酸酶B之醣化分佈以及醣化位置。
無論是逆相毛細液相層析或是短填充探針皆以C18顆粒當靜相並利用胜肽之疏水性（hydrophobicity）進行分離，而醣胜肽之分離主要靠其胜肽部分之疏水性質，因此直接將醣蛋白水解產物進行分離時，醣胜肽的層析訊號往往埋在眾多胜肽訊號之中，若醣胜肽與胜肽共溶析（coelute），則醣胜肽之質譜訊號會受到胜肽之抑制而使靈敏度下降，因此於分離前，利用纖維素微晶體將醣胜肽先進行純化，使醣胜肽之相對含量提高再進行分離，來避免胜肽之抑制，然而每經過一次純化，樣品的流失便越多，因此本研究嘗試以毛細管電泳直接分離經醣蛋白消化水解產物，利用胜肽與醣胜肽於電場下移動速度上之差異，來達到同時純化與分離的目的。本研究以毛細管電泳來分離醣蛋白經胰蛋白酶消化水解之胜肽與醣胜肽混合物，於酸性環境下，醣胜肽上的醣鏈對胜肽的等電點（isoelectric point）並無明顯貢獻，但大大增加了整體的分子大小，因此推測醣胜肽之電泳遷移率與未醣化之胜肽應有所差異，即可避免醣胜肽與胜肽共溶析（coelute）而造成醣胜肽之質譜訊號受到抑制，使醣蛋白水解產物無須經過醣胜肽純化之步驟，可直接以毛細電泳進行分離，並搭配第三章所發展之多次質譜/質譜方法來進行醣蛋白醣化分析。
生物體內醣蛋白之醣化並不都是固定的，某些醣蛋白之醣化會隨時間而改變，甚至與疾病相關。本研究最後引入同位素標記於醣胜肽上，期望對不同狀態下之醣蛋白之醣化進行相對定量，以瞭解不同狀態下醣化分佈之差異。在此嘗試了三種標記方式（1）18O之同位素標記：利用於H2O18中進行胰蛋白酶水解，便可標記上同位素之方法，但會受到胰蛋白酶進行水解事件之次數而影響到胜肽碳端標記上一個以及兩個18O之比例，進而影響到相對定量之結果，其相對定量準確度較還原甲基化法標記差，且反應時間相當長。（2）還原芐化法（reductive benzylation）標記：以胜肽標準品測試還原芐化法標記，其反應產率最高只能到達約80%，因此此標記不適合用於同位素標記之相對定量上；（3）還原甲基化法（reductive methylation）標記：此標記具有趨近完全之反應產率，無論以胜肽或是以醣蛋白：核糖核酸酶B之醣胜肽測試其同位素標記之相對定量，皆有相當高之準確性，之後預計將以還原甲基化法標記與層析做結合，以實際應用於醣蛋白醣化之相對定量分析。

關鍵字

質譜 ； 電灑法 ； 醣蛋白 ； 醣鏈結構 ； 醣化

並列摘要

Glycosylation is one of the most abundant post-translational modifications of protein. The carbohydrate chains of glycoproteins play a critical role in terms of protein structure and function. In this study, linear ion trap mass spectrometry was used for structure characterization of oligosaccharide and protein glycosylation analysis. For structure characterization of oligosaccharide, a method based on p-aminobenzoic ethyl ester (ABEE) closed-ring labeling and negative ion electrospray ionization tandem mass spectrometry is presented for linkage and branch determination for N-linked oligosaccharides cleaved from glycoprotein by Peptide-N-glycanase F. This approach was applied to two high mannose oligosaccharides M5G2, M6G2 cleaved from ribonuclease B and a complex oligosaccharide A2 cleaved from transferrin. In addition, this approach was also applied to determination of the ratio of isobaric glycan- M7G2 cleaved from ribonuclease B.
For protein glycosylation analysis, an approach for site-specific glycosylation analysis of glycoprotein has been developed. Glycoproteins were digested with specific protease, trypsin, yielding peptides and glycopeptides. Glycopeptides were enriched using cellulose microcrystalline and analyzed by reversed phase capillary liquid chromatography (CapLC) coupled with ESI linear ion trap mass spectrometry incorporating a full mass scan, and data-dependent multistage tandem MS. Because the MS/MS spectra of glycopeptides were dominated by fragmentations of glycosidic linkages, few information was provided on the peptide moieties. When the glycan moiety of glycopeptides is reduced to a single GlcNAc in MS2, MS3 analysis under data-dependent MSn setting could provide information on peptide sequence and glycan attachment site. This approach was applied to transferrin and bovine α-1 acid glycoprotein, glycosylation site as well as site-specific glycan profile were successful determined.
The procedures to carry out CapLC MS are time consuming, tedious, and carrying a potential for sample cross contamination. A simple, fast and low cost method has been developed for protein glycosylation analysis using a short C18 packing probe coupled with ESI tandem mass spectrometry. However, the peak width is too short to apply MS3 analysis, therefore, a method based on the rules of core structure for N-linked oligosaccharide and amino acid sequence for glycosylatioin site was developed for determining the glycosylation site using MS2 spectra. This strategy has been successfully applied to obtain the information of glycan profile and glycosylation site of ribonuclease B.
Glycoprotein was digested by trypsin and directly separated by capillary electrophoresis. Using capillary zone electrophoresis (CZE) under acidic condition, the mobility for glycopeptide is lower than peptide, and the co-eluting of glycopeptide with peptide could be largely reduced. Therefore, the step of glycopeptide enrichment could be eliminated and the overall sensitivity for glycosylation analysis was improved. The method was applied to a glycoprotein, transferrin, using CZE coupled with ESI linear ion trap mass spectrometry incorporating a full mass scan, and data-dependent multistage tandem MS; both glycosylation sites and site-specific glycan profile were successfully determined.
Glycosylation patterns can vary with development, regulatory state, type of disease and disease progression. Stable isotope labeling in combination with mass spectrometry was studied for relative quantification of protein glycosylation. Reductive benzylatioin, reductive methylation and 18O-labeling have been evaluated for their capability of relative quantification of protein glycosylation.

並列關鍵字

ion trap ； electrospray ； glycoprotein ； glycan ； glycosylation

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