功能化奈米粒子結合基質輔助雷射脫附游離飛行質譜儀應用於醣分子與醣基化胜肽之鑑定

Surface Functionalized Nanoparticles for Characterization of Glycans and Glycopeptides by MALDI-TOF MS

馬多娜(Indah Primadona)

指導教授 ： 陳玉如 ； 林俊成

國立清華大學/理學院/化學系/博士(2016年)

摘要

無機奈米粒子的特殊性質和它們與脈衝雷射光的交互作用，已經促使其應用在提升碳水化合物或醣類分子於基質輔助雷射脫附游離飛行質譜儀中的脫附游離作用。醣複合物普遍存在並於各種不同的生物反應中扮演著關鍵角色。由於其低穩定性和高度的結構多樣性，醣類結構鑑定通常需要進行化學衍生反應和 精密儀器的分析。然而，在大多數的情況中，衍生反應需要大量的樣品，因此提高了汙染物和副反應的風險。建立快速、靈敏、全面性的醣類定序方法並且無須進行化學衍生反應，依然是一個相當大的挑戰。
在本論文中，我們發展一個單步驟-虛擬串聯式質譜法的分析策略，利用此方法利用具有紫外線吸收特性的基質功能化磁性奈米粒子的和結合基質輔助雷射脫附游離質譜法建立分析平台。奈米粒子濃度調節的離子化和碎裂化方法以促進醣化合物的結構測定。省略了化學衍生步驟後，此分析策略能夠成功地分辨出三醣的異構物。低濃度的奈米基質增強了樣品中完整分子離子訊號，作為精確質量的判斷依據。另一方面，高濃度的奈米基質能促進廣泛和獨特的碎裂反應，包括高能鍵斷裂(A和X型交叉環列解)，這些特性有助於鑑定醣類的聯結和序列而無須利用常規使用的串聯質譜儀。此方法在分析複雜樣品上的可行性進一步於醣類混合物中進行評估，在此分析當中，分子離子能夠明確地偵測到，並且特徵產物離子提供足夠的特異性作為分子鑑定和異構物區別之用。除此之外，我們分析了多層奈米基質的各種作用:包含基質(能量吸收)、矽烷塗層(能量池和分散)、三氧化四鐵的核心(裂解作用)。基於光電能與熱能測量的結果，提出了奈米粒子引導游離及裂解中電子與能量的轉換機制。三醣的分辦測試作為初步奈米粒子輔助基質輔助雷射脫附游離質譜應用於醣類鑑定上，已經闡明了該方法策略的背後，部分奈米粒子中介的能量轉移動力學。這個結構專一性的質譜分子離子碎裂法，提供一個可用於確認未知物或闡明化學結構的有效的分析方法。
進一步，建立2,5-二羟基苯甲酸官能基化的碲化汞奈米粒子(HgTe@DHB NPs)作為基質輔助雷射脫附游離質譜中雙游離-脫附元件的分析方法，在單一檢測實驗中對複雜的醣類進行鑑別。以線性醣類為例，HgTe@DHB奈米粒子促進雷射誘導的廣泛分子解離反應，相較於HgTe微粒子和其他官能化或為官能化的無機奈米粒子(二氧化鈦，氧化鋅和三氧化二猛奈米粒子)，HgTe@DHB奈米粒子更增強目標醣類的偵測訊號。大量的特徵糖苷訊號(Y 和B型離子)和跨環裂解離子(A型離子)，使醣類的組成、序列、分支和不穩定的唾液酸化都能達成精確鑑定。此策略的廣泛應用性已藉由鑑定不穩定的唾液酸化醣類和兩組複雜的異構物醣類得到證實。我們的實驗結果證明，這個應用HgTe@DHB奈米粒子的虛擬串聯式質譜法，省略了化學衍生反應和常規的串聯式質譜分析法，有利於生物學相關和複雜醣類的分析。
最後，結合微波輔助去醣基化反應和奈米粒子輔助雷射脫附游離質譜，此簡易和創新的技術應用於醣胜肽的鑑定分析。從微波輔助去醣基化反應所產生的醣胜肽、醣基和胜肽，隨後以基質輔助雷射脫附游離質譜法進行分析。使用MNP@DHB作為基質，岩藻醣化 N型醣類複合物的組成、序列、分支和鏈結等資訊可被取得，而胜肽序列則以串聯式質譜儀技術偵測。此方法的適用性可藉由其可區分2種人工合成的N型醣胜肽異構物而證明。從觀察到的特殊糖苷鍵 (B3 ion, m/z = 536.8) 和跨環解離離子 (2,4A6, m/z = 1324.0)，終端具有岩藻醣基化的醣胜肽能明確地與核心岩藻醣基化的醣胜肽區分開來。
這項研究的成果有助於拓展分析化學的領域，以及推進奈米粒子輔助雷射脫附游離質譜法對醣類和醣類複合物偵測和鑑定的理解。)

關鍵字

納米粒子 ； 質譜 ； 基質輔助激光解吸/電離 ； 聚醣 ； 糖肽 ； 碎片

並列摘要

The unique properties of inorganic nanoparticles (NPs) and their interaction with pulsed laser irradiation have been exploited in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the enhanced desorption and ionization of carbohydrates or glycans. Glycoconjugates are ubiquitously present and play critical roles in various biological processes. Due to their low stability and incredibly high degree of structural diversity, the structural characterization of glycans generally requires chemical derivatization and sophisticated instrumentation. However, in most cases, the derivatization requires large sample amount and enhances the risk of introducing contaminants and side reactions. The development of rapid, sensitive and comprehensive glycan sequencing methods that do not require chemical derivatization remains a considerable challenge.
In this thesis, the development of single-step pseudo-MS/MS approach for tunable ionization and fragmentation to facilitate structure determination of glycoconjugates, using concentration-dependent UV-absorbing matrix-functionalized magnetic nanoparticles and MALDI MS was presented. Without chemical derivatization, this approach successfully distinguished isomeric sets of trisaccharides. Low concentration of nanomatrix provided enhanced signal for accurate mass determination of intact molecular ions in the sample. In contrast, high concentration of nanomatrix induced extensive and unique fragmentation, including high-energy facile bond breakage (A- and X-type cross-ring cleavages), which facilitated the linkage and sequence characterization of oligosaccharides without conventional tandem mass spectrometric instrumentation. The practicality of this approach for complex sample analysis was evaluated by an oligosaccharide mixture, wherein molecular ions are unambiguously observed and signature product ions are distinguishable enough for molecular identification and isomer differentiation. Subsequently, the roles of the multilayer nanomatrix components: matrix (energy absorption), silane-coating (energy pooling and dissipation) and core Fe3O4 (fragmentation) was also investigated. The plausible electron and energy transfer mechanism was proposed based on the threshold energy of photoelectrons and thermal energy measurements. The differentiation of tri-oligosaccharides, which served as the first step toward glycan characterization by nanoparticle-assisted MALDI-MS, had shed some insight on the nanoparticle-mediated energy transfer dynamics behind the proposed approach. The structure-specific fragmentation of molecular ions in mass spectrometry provides an efficient analytical method for confirming unknown analytes or for elucidating chemical structures.
Next, a method for complicated glycan characterization in a single assay by employing the 2,5-dihydroxybenzoic acid functionalized mercury telluride nanoparticles (HgTe@DHB NPs) as a dual ionization-dissociation element in MALDI-MS was developed. Using a linear glycan, HgTe@DHB NPs promote laser-induced extensive dissociation and intensive detection of the target glycan, superior to the HgTe microparticles and other functionalized and non-functionalized inorganic nanoparticles (TiO2, ZnO, and Mn2O3 NPs). Abundant generation of diagnostic glycosidic (Y-, and B-type ions) and cross-ring cleavage (A-type ion) ions permit unambiguous determination of the composition, sequence, branching, and linkage of labile sialylated glycans. The general utility of this approach was demonstrated on the characterization of labile sialylated glycans and two sets of complicated isomeric glycans. Our results show that this "pseudo-MS/MS” obtained by HgTe@DHB can be beneficial for the analysis of biologically relevant and more complicated glycans without the need of chemical pre-derivatization and conventional tandem mass spectrometry.
Lastly, a facile and innovative technique for glycopeptide characterization incorporating the microwave-assisted enzymatic deglycosylation and nanoparticle-assisted LDI-MS was designed and demonstrated. The mixture of glycopeptide, glycan, and peptide resulted from microwave-assisted deglycosylation reaction was subsequently analyzed in MALDI-MS. Using MNP@DHB matrix, the composition, sequence, branching, and linkage information of fucosylated N-glycoconjugates were acquired, while, the sequence of the peptide backbone was obtained by tandem mass spectrometry technique. The applicability of this method was shown by its ability to distinguish two synthetic isomeric N-glycopeptides. Through the observed unique glycosidic bond (B3 ion, m/z = 536.8) and cross-ring cleavage (2,4A6, m/z = 1324.0) ions, the terminal fucosylated glycopeptide can be unambiguously discriminated from core fucosylated glycopeptide.
It is expected that the results of this work can contribute to the ever expanding field of analytical chemistry, as well as advance the understanding in nanoparticle-based MALDI mass spectrometry for detection and characterization of glycans and glycoconjugates.

並列關鍵字

Nanoparticles ； Mass Spectrometry ； Matrix-assisted laser desorption/ionization ； Glycans ； Glycopeptides ； Fragmentation

參考文獻

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