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研究生: 魏怡安
Wei, I-An
論文名稱: 白蛋白和免疫球蛋白移除試劑對血清蛋白質體學分析的影響
Effects on Serum Protein Profile with Albumin/IgG Depletion Kit Treatment
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2016
畢業學年度: 104
語文別: 英文
論文頁數: 51
中文關鍵詞: 白蛋白和免疫球蛋白移除等電點聚焦分離方法equalizer 固定相共同移除蛋白質譜
英文關鍵詞: albumin and IgG depletion, solution-IEF, equalizer technology, co-depleted proteins, mass spectrometry
DOI URL: https://doi.org/10.6345/NTNU202203879
論文種類: 學術論文
相關次數: 點閱:84下載:3
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  • 血清為常見尋找生物標記物的來源,其中含有許多高含量的蛋白質,例如:白蛋白和免疫球蛋白。但由於高含量蛋白質於質譜分析中會遮蔽低含量蛋白質的訊號,我們通常會將其移除以利之後的分析。許多市售的親和性管柱可以有效的移除這些高含量的蛋白質,但使用的同時,部分和管柱沒有專一性的蛋白質也可能一併被移除,最終影響血清中蛋白質定量和定性的結果。本次實驗目的為使用Sigma 販售的ProteoPrep® 免疫親和性白蛋白和免疫球蛋白移除管柱,收集其濾液和沖提液搭配質譜分析技術,檢視經過移除高含量蛋白質的步驟對血清蛋白質體學分析的影響。我們將沖提液載入裝有Equalizer固定相的管柱,藉此降低高含量蛋白質與低含量蛋白質之間量的差異。另一分面,我們將濾液使用於等電點聚焦分離儀,藉此降低樣品的複雜度,並比較等電點聚焦分離方法運用於蛋白質層次與胜肽層次之解析度和所鑑定到的蛋白質個數。結果顯示,雖然兩者的解析度差異不大,但利用胜肽等電點聚焦分離方法可以多鑑定到54個蛋白質。血清蛋白質體學分析中,194個蛋白質鑑定於沖提液中而239個蛋白質鑑定於濾液中,最終有116個蛋白質同時被鑑定於兩者。為了確定經過免疫親和性管柱是否影響定量結果,我們挑選Complement C和 Alpha-2-Macroglobulin為例子,利用質譜多反應監測法進行定量分析。結果顯示,Complement C3在沖提液中的含量約占此蛋白質於血清中總量的百分之四左右,而Alpha-2-Macroglobulin在沖提液中的含量約占此蛋白質於血清中總量的百分之十左右。因此藉由本次實驗,我們發現經由移除高含量蛋白質的步驟,的確會影響血清中蛋白質的定性和定量的結果。

    Since albumin and IgG are two of the most abundant proteins in human serum, it is being removed in many MS-based assays for the detection of other low abundance proteins. By using commercially available kits, high abundance proteins in serum can be depleted efficiently. However, while removing these abundant proteins, it will also result in losing co-depleted proteins and possibly affecting the overall profile in quantitative proteomics. In this study, ProteoPrep® Immunoaffinity Albumin and IgG Depletion column (Sigma) was used; both elution and flow through fractions were preserved. Equalizer technology was applied on the elution fraction by removing the excess amount of high abundance proteins and contributed to concentrating the low abundance proteins. Besides, flow through fraction was divided into two portions. One was performed protein-level sIEF fractionation and the other was carried out peptide-level sIEF fractionation. Although the resolutions of two sIEF experiments were comparable, a total of 54 more proteins were identified in peptide-level sIEF fractionation. Serum proteome characterization with depletion kit treatment was achieved by making the comparison of protein identification between the elution fraction and the flow through fraction. One hundred and ninety four co-depleted proteins were identified and 116 of them were also detected in the flow through fraction. For example, Complement C3 and Alpha-2-Macroglobulin were two of the co-depleted proteins. By using MRM quantification approach, Complement C3 in the elution fraction was 4.3 % of its overall quantity and Alpha-2-Macroglobulin in the elution fraction was 10.5 % of its overall quantity. The result shows that with the immunodepletion procedure, proteins were nonspecifically co-depleted, leading to effects on protein quantitation and identification consequently.

    謝誌 i 中文摘要 iii ABSTRACT iv CONTENTS vi LIST OF FIGURES viii LIST OF TABLES x Chapter 1 Introduction 1 1.1 Human serum proteome 1 1.2 High abundance proteins depletion 2 1.3 Equalizer technology 3 1.4 Solution-IEF separation 4 1.5 Mass spectrometry 5 1.5.1 Electrospray ionization (ESI) 6 1.5.2 Tandem mass spectrometry 7 1.6 Protein identification 9 1.6.1 Peptide fragment fingerprinting (PFF) 9 1.7 Multiple reaction monitoring (MRM) 10 1.8 Purpose of this study 11 Chapter 2 Experiment section 12 2.1 Chemicals 12 2.2 Sample preparation and immunoaffinity depletion 12 2.3 SDS-PAGE 13 2.4 Equalizer technology 13 2.5 Desalting by reverse phase LC 14 2.6 Protein-level solution-IEF fractionation 14 2.7 Peptide-level solution-IEF fractionation 15 2.8 In-solution digestion 16 2.9 Analytical setups for LC-MS/MS 16 2.9.1 Waters Synapt G1 HDMS System 16 2.9.2 Orbitrap FusionTM TribridTM mass spectrometer 17 2.10 Protein identification 18 2.10.1 Data from Waters Synapt G1 HDMS System 18 2.10.2 Data from Orbitrap FusionTM TribridTM 18 2.11 Analytical setups for MRM 19 Chapter 3 Results and discussion 21 3.1 Serum Depletion 22 3.2 Equalization of bound proteins 24 3.2.1 Protein identification 25 3.3 Solution-IEF fractionation 27 3.3.1 Comparison of resolution 28 3.3.2 Protein identification 29 3.4 Serum protein profile 30 3.5 MRM analysis 31 Chapter 4 Conclusions 35 Reference 36

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