同位素標記電灑質譜法於不同哺乳期唾液酸寡醣變化之研究

Comparative Studies of Sialyloligosaccharides in Human Milk During Different Phases of Lactation Using Isotope Labeling and Electrospray Ion Trap Mass Spectrometry

劉希言(Hsi-Yen Liu)

指導教授 ： 何國榮

國立臺灣大學/理學院/化學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03300

摘要

母乳中唾液酸寡醣可抑制病原體，也是腦部發育的重要物質，對於新生兒生長相當重要。唾液酸寡醣的含量會隨著不同哺乳階段而有所改變，因此偵測母乳中唾液酸寡醣在不同哺乳階段中的相對變化，有助於對唾液酸寡醣生理功能之了解。
本研究利用甲基胺化法搭配同位素標記，將d0-和d3-甲胺標記3’-唾液酸乳糖及醣蛋白的醣鏈標準品，配製不同比例的混合溶液，純化後以電灑法質譜儀偵測，所獲得的訊號比例與配製比例做線性迴歸，得到斜率接近1，且線性相關係數(linear correlation coefficient, R2)也接近1，證明同位素甲基胺化法於醣鏈的相對定量具高可信度。使用此方法觀測不同哺乳階段母乳中七種唾液酸寡醣，的含量變化，發現隨著哺乳期增加，sialyllactose、sialyllactose-N-tetraose、monofucosyl,monosialyllacto-N-tetraose和disialyl-lacto-N-tetraose四種唾液酸寡醣的含量呈現先減少而後稍微回升的情況。而其餘三種sialyllacto-N-hexaose、monofucosyl,monosialyllacto-N-hexaose和difucosyl,monosialyllacto-N-hexaose唾液酸寡醣則先減少而後有1.5 ~ 2.3倍的增加。此外，利用二次質譜發現甲胺標記的3’-唾液酸乳糖和6’-唾液酸乳糖具有可區別之特徵碎片離子，藉由判定特徵碎片離子的相對強度，可同時對這兩種結構異構物相對定量，發現隨著哺乳期增加，3’-唾液酸乳糖含量減少18 ~ 38%，而6’-唾液酸乳糖含量幾乎維持不變。

關鍵字

質譜法 ； 穩定同位素標記 ； 甲基胺化法 ； 母乳 ； 唾液酸寡醣 ； 結構異構物

並列摘要

Sialyloligosaccharides of human milk are significant in anti-adhesion of pathogenic organism and the precursors for biosynthesis of brain for newborn infants. Accurate quantitative analysis of sialyloligosaccharides in human milk during different lactation phases is needed to investigate the physiological role of sialyloligosaccharides for newborn infants.
Methylamidation provides a simple and rapid labeling method for modifying the sialic acids of sialyloligosaccharides. Using d0/d3-methylamine stable isotopic labeling, methylamidation has been applied to relative quantitative analysis of sialyloligosaccharides by mass spectrometry. Applications of this method on 3’-sialyllactose and N-linked glycans released from transferrin demonstrates a 5-fold relative quantitative dynamic range, a satisfying linear dependency and slope between the molar ratios and the calculated intensity ratios of the d0/d3-derivatives. Comparative quantitative analysis for sialyloligosaccharides isolated from three different phases of lactation has been successfully performed by this technique. During lactation, the concentrations of sialyllactose, sialyllactose-N-tetraose, monofucosyl,monosialyllacto-N-tetraose and disialyl-lacto-N-tetraose are first decreased then increased slightly. The concentrations of sialyllacto-N-hexaose, monofucosyl,monosialyllacto-N-hexaose and difucosyl,monosialyllacto-N-hexaose are first decreased then increased by 1.5 ~ 2.3 folds.
In addition, by applying the proper collision energy, 3’-sialyllactose and 6’-sialyllactose can be differentiated by diagnostic peaks. Relative abundance analysis of each isomer can be achieved by comparing the intensities of the diagnostic peaks. We found that during lactation, the quantity of 3’-sialyllactose decreases by 18 ~ 38%, while that of 6’-sialyllactose remains the same level.

並列關鍵字

Mass spectrometry ； Stable isotopic labeling ； Methylamidation ； Human milk ； Sialyloligosaccharide ； Isomer

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