藉由鹵素燈誘發解離技術來增強醣類跨環碎片訊號在鑒定醣類分子結構之質譜分析應用

Enhancing Cross-Ring Fragmentation of Carbohydrate Molecules by Halogen Lamp-Induced Photodissociation to Assist the Structural Analysis in Mass Spectrometry.

黃培桓(Pei-Huan Huang)

指導教授 ： 吳志哲

共同指導教授 ： 王亦生(Yi-Sheng Wang)

國立暨南國際大學/科技學院/應用化學學系/碩士(2021年)

https://doi.org/10.6837/ncnu202100076

摘要

質譜技術在應用於醣類分子的結構解析上，需要先將醣類分子進行裂解，來產生足夠的醣苷鍵及環內斷鍵碎片資訊。本論文開發了一套適用於醣類分子結構解析的鹵素燈源光裂解技術，用來增加醣類分子的裂解碎片資訊，特別是環內斷鍵碎片資訊。本論文所使用的鹵素燈源光裂解 (Halogen Lamp Photodissociation，HLP)技術相較於傳統的醣類分子裂解方法相比，能夠產生更多的環內斷鍵碎片資訊。將此開發的醣類裂解方法與一般商業化質譜儀搭載的離子源後裂解(Post Source Decay, PSD)方法相比較後，發現HLP與PSD裂解方法在醣類結構的斷鍵上會產生不同的裂解模式偏好。PSD模式的碎片大多為醣苷鍵的斷鍵碎片，HLP模式則產生更多的環內斷鍵碎片資訊。在醣類結構分析中，這些環內斷鍵的碎片提供了更多醣類分子組成結構的資訊。本實驗也將這套鹵素燈源光裂解技術成功地 應用於分辨兩種葡聚醣的同分異構物與三種結構相似的肝素雙醣。
本篇論文透過自組裝的鹵素燈杯燈架的光學系統連續以紅外光連續對醣類分子與基質的共結晶混合物進行照攝3.5小時後，將樣品進行基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF-MS)質譜分析，再透過醣類碎片組成模擬軟體(GlycoWorkbench)對實驗圖譜進行碎片比對。在鹵素燈光裂解作用之後，經過光處理過後的醣類分子，幾乎都能提供每個單醣上的環內斷鍵資訊。在比起未照光作用之前，所有可能的碎片數量增加10-30%左右。在異麥芽三醣(Isomaltotriose , IM3)樣品的光裂解碎片圖譜分析中，實驗結果能夠比對出九成模擬軟體所列出的碎片。
本篇論文將兩個六聚醣分子的同分異構物，在經過鹵素燈源光裂解處理過後，經質譜分析後發現六聚醣分子的同分異構物之間，各自具有高特異性的質譜峰值訊號，透過這些特徵峰值碎片，我們可以對六聚醣分子的同分異構物進行區分。我們也嘗試將此鹵素燈源光裂解應用在分辨具相同分子量的肝素雙醣結構異構物上，也在質譜圖譜上發現出各自對應的特徵峰值碎片，為質譜分析應用於解析肝素雙醣結構異構物上提供一套有力的分析工具。
本論文所提出的鹵素燈源光裂解技術，僅須透過簡單的儀器架構裝置，即可將此方法結合基質輔助雷射脫附游離飛行時間質譜儀進行樣品分析。本篇實驗驗證此方法可用於多種醣類樣品的結構分析，並具有不需要透過串聯質譜分析的優勢。對於醣類的結構分析來說，是個具有優勢以及前景的醣類裂解方法。

關鍵字

醣類結構 ； 鹵素燈源光裂解 ； 源內衰變 ； 環內斷鍵 ； 醣苷鍵斷鍵

並列摘要

Mass spectrometry has been widely used for structural analysis of carbohydrate molecules. In MS analysis, carbohydrate molecules need to be fragmented in order to generate enough glycosidic bonds and cross-ring fragments for comprehensive structural identification. In this thesis, a halogen lamp-based photodissociation (HLP) system is used for enhancing the cross-ring fragmentation of carbohydrate structures.
In this work, a mixture of carbohydrate molecules and organic matrix spotted on the MALDI sample plate is illuminated with halogen lamp light for 3.5 hours at an power of 21.6 W/cm2, and then the sample is subjected to MALDI-TOF MS analysis. The resulting mass spectra were analyzed using Glycoworkbench software.
After HLP pretreatment, the number of identified fragment ions can be increased by 10 to 30%. The number of fragment can be identified by this method is up to 50%-70%. We applied this new method for the structural analysis of Globo-H, Isomaltotriose (IM3), Isomaltohexaose, and heparin disaccharides. In comparison to conventional Post Source Decay (PSD) fragmentation method, the total number of fragments derived from the HLP method is significantly higher than that derived from the PSD method, especially in the number of cross-ring fragments. The majority of the fragments from the PSD mode are attributed to the fragmentation of glycosidic bonds. The HLP method was further applied to distinguish two structural isomers of oligosaccharides composed of six carbohydrate molecules (isomaltotriose and laminarihexaose,) and three structural isomers of heparin disaccharides. We found that each structural isomer exhibits distinct MS fragmentation patterns for structural identification after the HLP treatment.
The halogen lamp-assisted photodissociation method is a simple but effective ambient fragmentation method for enhancing the fragmentation of carbohydrate molecules. It can be used in conjunction with the MALDI-TOF MS for structure identification, without the use of tandem mass spectrometry. We had successfully applied it to analyze various carbohydrate structures including two types of structure isomers. This is a new and promising tool for the structure analysis of carbohydrates.

並列關鍵字

carbohydrate structure ； halogen lamp photodissociation ； post source dacay ； cross-ring fragment ； glycosidic fragment

參考文獻

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