眼鏡蛇咬傷在非洲和亞洲的農村地區是常見的醫學問題，被咬傷的受害者會引起神經毒性和局部組織損傷。這種組織破壞特性大多與高度豐富的眼鏡蛇毒細胞毒素有關。有趣的是，一些眼鏡蛇不具備這種細胞毒性屬性，反而有些眼鏡蛇更依賴神經毒素。然而，有另一組蛋白質，叫做高分子量蛋白質(HMW)大量地存在大多數蝰蛇毒液中，它們會引起局部效應，如組織壞死。然而，在眼鏡蛇毒液中，高分子量蛋白質的功能沒有得到適當的闡明，因為它們被認為不會導致主要的蛇咬傷症狀。但是，我們的小組認為眼鏡蛇毒液高分子量蛋白質必須在毒液中發揮重要作用，因此，適當的特徵是必要的。在傳統的分離方法中，高分子量蛋白質由於其分子大小而難以量化。於是，我們首次用以奈升級液相層析串聯式質譜學對於蛇毒進行調查。六種眼鏡蛇毒液(Naja atra, kaouthia, nivea, philippinensis, sputatrix 和Hemachatus haemachatus)的MS/MS結果與兩種不同的定量演算法進行了比較：Top3和iBAQ。我們也采用限制性和審查的眼鏡蛇毒液蛋白資料庫進行定量。Top3 與以前的相同毒液樣本研究相比，表現出更合理的豐度和更高的相關性。我們確定了在六種眼鏡蛇毒液裏含有豐富的三指毒素，並建立了相當數量的高分子量蛋白質，可分別達到 Top3 和 iBAQ 重量百分比的
20% 或 40%。這種定量方法快速量測眼鏡蛇毒液，而且只需要低毒液樣品，降低了分析成本。因此，我們提出了以奈升級液相層析串聯式質譜學和Top3的演算法，用於未來高分子量蛋白質的識別和定量需求。

Cobra bites are a common medical issue in rural parts of Africa and Asia where they cause neurotoxicity and local tissue damage in bitten victims. This tissue destroying property is mostly associated with highly abundant cobra venom cytotoxins. Interestingly, some cobras do not possess this cytotoxic attribute and rely more on neurotoxins. Another group of proteins, high molecular weight proteins (HMW), are present vastly in most viper venoms where they cause local effects such as tissue necrosis. In cobra venom however, their function has not been properly elucidated because they are thought to not contribute to major snakebite symptoms. Nevertheless, our group believes that cobra venom HMW proteins must have an important role in envenomation and therefore proper characterisation is necessary. But in conventional separation methods, HMW proteins are poorly quantified due to their size. Here, we investigate for the first time the use of nanoflow liquid-chromatography coupled with tandem mass spectrometry (nLC-MS/MS) for snake venomics. The MS/MS results of six cobra venoms (Naja atra, kaouthia, nivea, philippinensis, sputatrix and Hemachatus haemachatus) were compared with two different quantification algorithms: Top3 and iBAQ. A restricted and reviewed cobra venom protein database was used for quantification. Top3 showed more reasonable abundances and had higher correlation compared to previous studies with the same venom samples. We determined an abundance of three-finger toxins and established a significant higher amount of HMW proteins that could reach up to 20% or 40% of weight percentage for Top3 and iBAQ, respectively. This approach is fast and low venom sample is required, which decreases the cost of analysis. We therefore propose nLC-MS/MS and the Top3 algorithm for future need in identification and quantification of HMW proteins.

ABBREVIATIONS --------------------------------------------1
Chapter 1 Introduction -------------------------------------3
1.1 Snakebites and cobras ----------------------------------3
1.2 Cobra venom proteins and function cobras ------------4
1.3 Venom proteome determination and significance------7
Chapter 2 Material and methods ---------------------------10
2.1 Venom samples -----------------------------------------10
2.2 Nanoflow LC separation --------------------------------10
2.3 MS/MS analysis -----------------------------------------11
2.4 Construction of sequence database --------------------12
2.5 Quantitative data analysis ------------------------------ 12
2.6 PLA2 activity -------------------------------------------- 13
2.7 Statistical analysis ---------------------------------------14
2.8 Phylogenetic sequence analysis ------------------------14
2.9 Sequence alignment ------------------------------------14
Chapter 3 Results -------------------------------------------15
3.1 Venomics of six cobra venoms with Top3 and iBAQ ---15
3.2 Comparison with the traditional approach -------------16
3.3 HMW protein quantification and evolution ------------17
Chapter 4 Discussion ---------------------------------------35
Conclusion --------------------------------------------------39
References --------------------------------------------------40
Appendix ---------------------------------------------------48

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