同位素標記電灑法質譜與串聯質譜於醣蛋白及牛奶寡醣變化之研究

Comparative Study of Glycoproteins and Milk Oligosaccharides Using Isotope Labeling and Electrospray Linear Ion Trap MS and Tandem MS

馬義鈞(Yi-Chun Ma)

指導教授 ： 何國榮

國立臺灣大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.01103

摘要

實驗室先前發表了完整醣胜肽的相對定量策略，可以判定兩核醣核酸酶B樣品醣蛋白濃度、醣鏈分佈、醣化程度的變化。然而有多個醣化位置和複雜型醣鏈的醣蛋白更為常見，如果此相對定量策略可以應用於有多個醣化位置和複雜型醣鏈的醣蛋白，將會對醣蛋白分析非常有幫助。與之前的策略相比，此策略使用液相層析質譜、質量差較大的同位素標記(6 Da)及自然同位素貢獻的訊號修正。兩胰蛋白酶水解醣蛋白樣品分別與12C, H-甲醛和13C, D-甲醛反應標記2CH2和213CD2，混和後以纖維素微晶體純化，以液相層析質譜分析。從非醣化胜肽的2CH2和213CD2標記訊號可以得到醣蛋白濃度變化，比較醣化位置上2CH2和213CD2的醣鏈分佈可以得到醣鏈分佈變化，得到醣蛋白濃度變化與醣鏈分佈變化後，可以計算此醣化位置的醣化程度變化。配置兩個醣蛋白濃度(2倍變化)與醣化程度不同(100%與25%醣化程度)但醣鏈分佈相同，及兩個醣蛋白濃度與醣化程度相同但醣鏈分佈不同(原始的與β1-4 galactosidase處理的醣鏈分佈)的去唾液酸胎球蛋白樣品。對於唾液酸胎球蛋白，則配置兩個醣蛋白濃度(4倍變化)、醣化程度(100%與50%醣化程度)與醣鏈分佈(原始的與溫和酸性條件處理的醣鏈分佈)皆不同的樣品。結果發現得到的醣蛋白濃度變化、醣鏈分佈變化與醣化程度變化非常接近配置值。因此，此策略將可以應用於大部分醣蛋白的相對定量分析。
為了提升醣胜肽在質譜分析的感度，並進行準確的相對定量，在本研究中，開發一策略使用同位素標記及離子阱二次質譜，對醣胜肽進行相對定量。由於標記在胜肽端，因此為了進行相對定量，二次質譜偵測的碎片離子必須包含胜肽部分，在此策略中，運用離子阱二次質譜可以為醣胜肽帶來強度很高的包含胜肽端的碎片的優點，因此與三段四極柱或是三段四極柱-離子阱質譜儀相比，將可以帶來更好的感度。在離子阱二次質譜偵測時，偵測的碎片離子為胜肽加一個GlcNAc與另兩個強度最高的包含胜肽端的碎片離子。結果發現使用二次質譜分析去唾液酸胎球蛋白與唾液酸胎球蛋白醣胜肽與一次質譜相比，得到了1.6 - 52.9倍S/N提升。使用二次質譜偵測2CH2及213CD2標記去唾液酸胎球蛋白與唾液酸胎球蛋白的胰蛋白酶水解混和物，由2CH2及213CD2標記碎片離子的訊號，可以得到非常接近配置的輕與重標記濃度比例的結果。由以上結果，證實同位素標記與離子阱二次質譜可以提供醣胜肽靈敏並且準確的相對定量方法。
牛乳寡醣是非常複雜的同分異構物混和物，寡醣同分異構物其單糖組成相同只差在單糖鍵結點不同，然而其異構物在新生兒腸道內的生理功能可能完全不同。在本研究中，使用實驗室先前發表的對胺基苯甲酸乙脂(ABEE)閉環標記，來分析牛乳寡醣同分異構物。將牛乳寡醣純化後，閉環標記ABEE，將衍生物再經純化後，以C18毛細液相層析負離子串聯質譜分析ABEE標記寡醣。使用ABEE標記寡醣的特徵裂解離子偵測寡醣，因此對於異構物的分析，除了可以使用液相層析將時間分開，也可以使用串聯質譜將質量分開。在本研究中取得了牛分娩1 - 4天的牛初乳，將1 - 4天初乳各加入Glc4標準品，分析Glc4訊號得到前處理方法的再現性約8%，並對牛奶中含量豐富且報導有生理功能的寡醣異構物包括3GalNL、6GlcNL、LNT、LNnT、LNnH、3SL、6SL、3SLN、6SLN、3S6GL、6S3GL及DSL進行哺乳期的含量分析。其中，LNT在哺乳期間的含量變化為第一次在本研究中被報導。

關鍵字

醣胜肽複雜型醣鏈 ； 醣蛋白濃度 ； 醣鏈分佈 ； 醣化程度 ； 相對定量 ； 二維離子阱 ； 電灑法質譜 ； 串聯質譜 ； 牛初乳 ； 對胺基苯甲酸乙脂 ； 閉環標記

並列摘要

A comparative strategy has been demonstrated using RNase B, a single-site N-linked high-mannose glycoprotein. Glycoproteins are more common with multiple glycosylation sites and with complex glycans. A strategy capable of differentiating the changes caused by glycoprotein concentration, glycosylation site occupancy, and a glycoform profile of complex glycoproteins would be beneficial. Compared to our previous approach, we made several modifications including the use of RPLC, a larger mass difference isotope tag, and isotope overlapping correction. Tryptic-digested glycoproteins were labeled using 12C, H-formaldehyde and 13C, D-formaldehyde, purified, and then analyzed using capillary RPLC-MS. The relative intensity of non-glycosylated peptides provided information on glycoprotein concentration variation. A site-specific glycoform profile variation was obtained by comparing the glycoform profile of CH2 and 13CD2 glycopeptides. Determining the protein concentration and glycoform profile variations allows the glycosylation site occupancy variation to be calculated. A strong correlation between the observed and prepared ratios for asialofetuin and fetuin glycopeptides from 0.2 to 5 was obtained. Two asialofetuin samples with either different glycoprotein concentrations (2-fold change) and glycosylation site occupancies (100% and 25%) but the sample glycoform profile, or the same glycoprotein concentration and glycosylation site occupancy but different glycoform profiles (normal and modified) were prepared. Two fetuin samples with different glycoprotein concentrations (4-fold change), glycoform profiles (normal and modified), and glycosylation site occupancies (100% and 50%) were prepared. Samples were labeled, mixed, purified, and analyzed using RPLC-MS. The results of the comparative study on asialofetuin and fetuin had a strong correlation with the prepared values. The modified approach is expected to be applicable to most glycoproteins.
To improve the glycopeptide detection in a stable isotope labeling quantification method, a strategy was proposed using MS/MS analyisis on an ion trap mass spectrometer of abundant glycan fragmented ions which contain the labeled peptide moeity. The peptide plus the innermost GlcNAc and the other two most abundant glycan fragmented ions which contain the peptide moiety were monitored. A 1.6- to 52.9-fold S/N improvement was obtained for asialofetuin and fetuin tryptic glycopeptides. Heavy (213CD2) and light (2CH2) labeled asialofetuin or fetuin tryptic digest were mixed and analyzed respectively. The calculated heavy to light ratios based on 2CH2 and 213CD2 labeled MS/MS fragment ions for 6 and 17 asialofetuin and fetuin glycopeptides, respectively, were very close to the prepared ratio. The results showed that stable isotope labeling and ion trap MS/MS could provide a sensitive and accurate glycopeptide relative quantification method.
Bovine milk oligosaccharide is a mixture of isomeric oligosaccharide structures. The oligosaccharide isomers could have distinct biological functions in the gastrointestinal tract of the newborns. A strategy was proposed to analyze bovine milk oligosaccharide isomers using ABEE closed-ring labeling and negative ion electrospray tandem MS. Bovine colostrum oligosaccharides were isolated, closed-ring labeled with ABEE, purified, and analyzed using C18 capillary liquid chromatography negative ion electrospray tandem MS. ABEE labeled oligosaccharides were detected using the linkage specific fragments, therefore isomeric structures were not only separated based on retention time using HPLC but also m/z using tandem MS. Analytical variation for sample preparation procedure was estimated to be 8% by analyzing the spiked Glc4 standard to bovine milk collected from 1 - 4 day postpartum. Oligosaccharide variation in 1 - 4 day postpartum was studied. For the first time the variation of LNT along bovine lactation period was established in this study.

並列關鍵字

glycopeptide ； complex-type glycan ； glycoprotein concentration ； glycan profile ； glycosylation site occupancy ； relative quantification ； linear ion trap ； electrospray mass spectrometry ； tandem mass spectrometry ； bovine colostrum ； p-aminobenzoic acid ethyl ester ； closed-ring labeling

參考文獻

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