不同結構的醣類具有不同的功能性，因此若能深入分析各種醣類的表徵以及結構，有助於了解醣類對於健康以及各種疾病的影響和重要性。儘管使用基質輔助雷射脫附游離法質譜儀可以擁有高靈敏度且快速和高通量分析，由於醣類結構相似性和複雜性較高以及游離化效率較低，導致在偵測上難以分離和鑑定。因此，本實驗主要利用薄層層析分離技術線上結合基質輔助雷射脫附游離法質譜儀，並且藉由離子液體來穩定以及均勻分散奈米粒子基質，以開發可以快速分離和同時鑑定寡糖結構有效的剖析平台。首先，在薄層層析方面選擇合適的展開溶劑以及選擇適合分離寡醣化合物的薄層層析種類。再者，發展奈米基質的沉積方法和找尋適合均勻分散二羥基苯甲酸之磁性奈米基質(DHB@MNP)的溶劑，以達到在薄層層析上，可以一次步驟游離及裂解而能有效率鑑定醣類化合物之結構。第三，找尋薄層層析線上連接基質輔助雷射脫附游離法質譜儀最佳化的收集圖譜的參數。在此實驗中，若要將二羥基苯甲酸之磁性奈米基質(DHB@MNP)旋轉塗佈薄層層析上，須使用離子液體當作溶劑，其可以幫助基質均勻分散在薄層層析上，除了可以提高訊號的再現性(<20%CV)同時又可以增加訊號12和28倍的靈敏度。而相較於正相薄層層析，逆相的薄層層析對醣類混合物可以擁有更好的分離效果以及增加12倍訊號的靈敏度。另外，薄層層析已經將大部分寡醣分離，但是對於同分異構的寡醣卻無法分離，然而在此實驗系統中，利用磁性奈米基質所產生內能轉換而造成醣類生成診斷性的糖苷和交叉環裂解離子，從而能夠及時對每個分離的斑點進行聚醣的組成，序列，分支和連接的結構解析。
人乳寡糖（HMO）是生物活性成分，可調節嬰兒的免疫功能，並通過建立腸道菌群和免疫系統為新生兒提供多層保護。因此了解寡糖的結構功能，能有助於在人工營養中設計具有抗感染性的新型治療劑。所以在本論文中的第二部分，我們將此開發的平台應用在分析不同供應者在不同泌乳時間寡醣含量及種類的鑑定。相較於傳統耗時的分析方法，例如需要將醣類樣品衍生化，以及將薄層層析顯色或者是使用串聯式質譜儀，本實驗設計的分析平台可以快速鑑定人乳中的25種寡糖。 藉由使用人乳寡醣的相對訊號強度進行定量分析，並且根據熱圖分析顯示，可以得知在不同泌乳時間和各個供應者的樣品中寡糖含量的差異。 在供應者A和B的樣品（5-12天）中觀察到更多更大的寡醣比在供應者E的樣品（12個月）中更高。 這種差異性可以提供有關嬰兒的微生物群和免疫力的相關資訊。 隨著我們樣品製備方法的簡單性以及良好的分離效果和能鑑定的結構表徵，我們的方法可用於含量較低的寡醣樣品的快速篩選。

The in-depth characterization of glycan structure is crucial to understanding their structure-function relationships and their effects on health and various diseases. Despite advances in rapid analysis, the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is limited for complex mixture of carbohydrates due to their low ionization efficiency and the difficulty in separating oligosaccharides because of their high structural similarity. In the first part of the thesis. We developed an efficient oligosaccharides profiling approach based on ionic liquid (IL)-stabilized, nanomatrix-decorated, thin-layer chromatography (TLC)-MALDI MS for simultaneous and rapid separation, detection and identification of oligosaccharides. First, optimization of TLC developing solvent systems and selection of appropriate plate nature for separation of saccharides mixture. Second, evaluation of nanomatrix deposition methods and an efficient dispersant selection for utilizing dihydroxybenzoic acid-functionalized magnetic nanoparticles (DHB@MNPs) nanomatrix for a single step ionization and fragmentation of carbohydrates directly from the TLC surface. Third, selection and parameter tuning of TLC-MALDI MS coupling methods and spectra acquisition modes. The IL demonstrated good dispersion and stabilization for the spin coating of DHB@MNPs on the TLC plate with spot homogeneity, which contributed to the observed high reproducibility (<20% CV) and 12- and 28-fold signal enhancement. In addition, compared to normal phase (NP) TLC, C18 modified reverse phase (RP)-TLC showed superior separation and 12-fold signal enhancement for mixture carbohydrates. Though the TLC was limited to separate isomeric glycans, the IL-dispersed DHB@MNPs facilitate unique high-energy bond breakage to generate diagnostic glycosidic and cross-ring cleavage ions, enabling unambiguous elucidation of the isomeric pairs of Lewis oligosaccharides. DHB@MNP-assisted fragmentation enables on-spot structural elucidation of composition, sequence, branching, and linkage of glycans in each separated spot.
Human milk oligosaccharides (HMOs) are bioactive components that modulate the immune function of the infant and give multiple layers of protection for newborn by establishing gut flora and immune system. However, understanding oligosaccharide structure-function is very essential to design a novel therapeutic agents with anti-infection in artificial nutrition. Thus, in the second part of the thesis, this approach was applied to HMO analysis of the samples collected from donors at different lactation times. Without chemical derivatization of glycan samples, glycan visualization by TLC and tandem MS, our integrated platform, allowed the identification of 25 oligosaccharides from human milk. By quantitative analysis using the relative abundance of each HMO, heatmap analysis revealed the variability of the oligosaccharide in samples from individual donors taken at different periods of lactation time. Higher numbers of larger oligosaccharides were observed in the samples from donors A and B (5-12 days) than in that from donor E (12 months). Such variation may provide insight for further study of the microbiota and immunity of infants. With the demonstrated simplicity of our sample preparation method along with the achieved separation and in-depth structural characterization, our approach can be used for the rapid screening of other oligosaccharide-rich samples.

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