運用兩性親水作用層析磁性奈米粒子順序性純化醣基化和磷酸化胜?

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陳昱瑋(Yu-Wei Chen) 查詢紙本館藏

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化學學系

論文名稱

運用兩性親水作用層析磁性奈米粒子順序性純化醣基化和磷酸化胜?
(Sequential Enrichment of Glycopeptides and Phosphopeptides by Zwitterionic-HILIC (ZIC-HILIC) Magnetic Nanoparticles)

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摘要(中)

在多種蛋白質轉譯後修飾中，N-連接醣基化和磷酸化在生物功能上扮演著相當重要的功能，像是細胞訊息傳導、蛋白質的穩定性和移動。然而，由於固有的修飾異質性和質譜游離效率的相互抑制，因此發展醣基化和磷酸化胜?純化的方法是必須的。雖然已有不少純化方法針對這兩種修飾，但多數方法都是針對兩種轉譯後修飾個別分析，近來，磁性奈米粒由於其能快速分離的能力和表面易於進行修飾而被廣泛地運用。
在此論文工作中，基於親水性作用力、活化的胺基和兩性離子官能基被修飾在磁性奈米粒子(由Elmer Austria Jr.合成並提供)的表面上用以純化出磷酸化和醣基化胜?。藉由實驗條件的最佳化後，包含溶液組成、pH值、奈米粒子使用量和有機相比例，在醣基化胜?的純化中，0.1%甲酸/95%乙?(調整至pH7)作為活化緩衝液，而1%磷酸/80%乙?和0.1%氨水/80%乙?則作為洗滌緩衝液，最後由2%氨水/50%乙?作為析出緩衝液。另外在磷酸化胜?的純化方面，0.1%甲酸/95%乙?(調整至pH 3) 作為活化緩衝液，而6%乙酸/85%乙?
則作為洗滌緩衝液，最後由5%氨水/50%乙?作為析出緩衝液。在磁性奈米粒子用量方面，醣基化/磷酸化胜?的純化皆是以2微克胜?比上100微克磁性奈米粒子的用量為最佳化條件。此外，藉由配合調整酸鹼值的策略，可在為α-和 β-酪蛋白(α-, β-casein)(磷酸化胜?)以及辣根過氧化物?(醣基化胜?)1:1的混和胜?中，在使用基質輔助雷射脫附游離飛行質譜儀的分析下，有效地在pH值3的環境下萃取分離出10條磷酸化胜?以及在pH 7的環境下得到9條醣基化胜?，分離效率分別為96.3% 和81.1%。
為增加樣品複雜度，在樣品中添加了牛血清白蛋白。在α-和 β-酪蛋白、辣根過氧化物?和牛血清白蛋白1:1:1:1的混合胜?樣品中，8條磷酸化胜?和7條醣基化胜?能分別在不同的沖提順序下被鑑定到。當提高了牛血清白蛋白的比例時，仍然有8條磷酸化胜?被鑑定，但卻分別只有4條和5條醣基化胜?在1:1:1:5和1:1:1:10的比例中被鑑定到。
接著將方法應用在PC9非小型細胞肺癌細胞(non-small-cell lung cancer cell)中，藉由直接純化醣基化胜?的方式，鑑定到306條醣胜?和35種醣型，此外，在去醣基化的作用下，1068條去醯胺基的胜?被鑑定到。另一方面，246條磷酸化胜?(231條單一磷酸化,15條多重磷酸化)則在直接純化磷酸化胜?被偵測到。在起始200微克的PC9膜胜?，經過除鹽和順序性純化策略下，246條磷酸化胜?(209條單一磷酸化, 27條多重磷酸化),432條去醯胺基的胜?以及243條醣胜?(24種不同的醣型)皆在液相層析串聯三合一式質譜儀的分析下被鑑定。
這些結果都顯示，此種兩性親水作用磁性奈米粒子在針對大範圍的醣基化和磷酸化胜?的純化和鑑定上，都具有相當高的發展潛力。

摘要(英)

Among various protein post-translational modification (PTM), N-linked glycosylation and phosphorylation are among the most ubiquitous PTMs and play important roles in many biological functions, such as signal transduction, protein stability, and mobility. However, due to inherent heterogeneity of modification site and structure and ion suppression effect from free peptides during mass spectrometry analysis, it is necessary to develop enrichment strategy prior to mass spectrometry (MS) analysis. Though there are many enrichment methods developed for these two PTMs, both glycopeptides and phosphopeptides require different enrichment approaches.
Recently, magnetic nanoparticles (MNPs) have been widely used in biomedical applications mainly because of its capability for surface functionalization and fast separation by magnetic extraction. In this thesis, based on the hydrophilic interaction, active NH2- and zwitterionic functional groups were anchored (provided by Elmer Austria Jr.) on the surface of the magnetic nanoparticles (amine/zwitterionic-hydrophilic interaction based magnetic nanoparticle, NH2/ZIC-HILIC MNP) for simultaneous enrichment of glycopeptides and phosphopeptides. Four main parts were optimized in this work, including buffer composition, pH value, organic composition and particle amount. In glycopeptide enrichment, 0.1% formic acid (FA) with 95% acetonitrile (ACN) (adjusted to pH 7) were used as incubation buffer; 1% phosphoric acid with 80% ACN and 0.1% ammonium hydroxide (NH4OH) with 80% ACN were applied as washing buffer and 2% NH4OH/50% ACN for elution buffer respectively.
On the other hand, 0.1% FA/95% ACN (adjusted to pH 3) were used as incubation buffer. 6% acetic acid (AA) with 85% ACN and 5% NH4OH/50% ACN were applied as washing buffer and elution buffer respectively. 2μg peptides to 100μg nanoparticles was the optimized usage ratio for both glycopeptide and phosphopeptide enrichment.
By pH modulation strategy, effective separation of both glycopeptides in pH=7 and phosphopeptides in pH=3 can be achieved from a 1:1:1 mixture of α- and β-casein (phosphoproteins) and HRP (glycoprotein), 10 phosphopeptides in phosphopeptide elute fraction and 9 glycopeptides in glycopeptide elute fraction were identified in MALDI-TOF analysis with 96.3% and 81.1% separation efficiency for phosphopeptides and glycopeptides respectively.
To increase the complexity of the sample, bovine serum albumin (BSA) protein was added into the sample. In ratio of HRP : α-casein : β-casein : BSA= 1:1:1:1, 8 phosphopeptides still can be detected and dominate in the phosphopeptide elute fraction; in the glycopeptide elute fraction, though there are still 7 glycopeptides can be detected, When BSA ratio go higher up to 1:1:1:10, no significant influence in phosphopeptide fraction, although still 8 phosphopeptides were still detected, however, there are only 4 and 5 glycopeptides were detected in ratio 1:1:1:5 and 1:1:1:10 respectively.
Further application to the proteome level was demonstrated in PC9 cell line, a non-small cell lung cancer (NSCLC) cell line. By direct glycopeptide enrichment, 306 unique intact glycopeptides and 35 unique glycan structure were identified. With deglycosylation treatment by PNGase F, 1068 deamidated peptides with consensus motif NxS/T/V/C (x belongs to any amino acids but not proline) were detected. On the other hand, 246 phosphopeptides (231 mono-phosphorylated and 15 multi-phosphorylated) were identified in direct phosphopeptides. With initial 200μg membrane peptide from PC9 cell and followed by desalting and sequential enrichment strategy, 236 phosphopeptides (209 mono-phosphorylated and 27 multi-phosphorylated) were detected in phosphopeptide elute fraction. 243 unique intact glycopeptides (24 unique glycan structure) and 432 deamidated peptides with glycosylation motif (after deglycosylation) were identified in glycopeptide elute fraction combined with LC-MS/MS analysis using orbitrap fusion lumos mass spectrometry. These results further demonstrate that NH2/ZIC HILIC MNP can be a highly-potential method for characterization of both phosphopeptides and glycopeptides.

關鍵字(中)

★ 磷酸化
★ 醣基化
★ 磁性奈米粒子
★ 兩性親水層析作用

關鍵字(英)

★ phosphorylation
★ glycosylation
★ magnetic nanoparticle
★ Zwitterionic-HILIC

論文目次

中文摘要 i
Abstract iii
誌謝 vi
Table of Context vii
List of Abbreviations x
Chapter 1 Introduction 1
1.1 Protein glycosylation 1
1.1.1 Analyzing glycosylation in proteins by mass spectrometry approach 2
1.2 Protein phosphorylation 9
1.2.1 Analyzing phosphorylation in proteins 10
1.3 Magnetic nanoparticles (MNPs) 12
1.3.1 Enrichment of glycopeptides and phosphopeptides by using MNPs 13
1.4 Thesis Organization and Objectives 15
Chapter 2 Material and Methods 17
2.1 Materials 17
2.2 Method 18
2.2.1 Preparation of magnetic of nanoparticle 18
(1) Fabrication of core MNP via thermal decomposition method 18
(2) Ligand encapsulation by using amidosulfobetaine-14 (ASB-14) 19
(3) Silica and amine functionalization of the MNPs 19
2.2.2 In-solution digestion for standard proteins 20
2.2.3 Membrane protein extraction 21
2.2.4 Gel-assisted Digestion 22
2.2.5 Desalting (SDB-XC) 24
2.2.6 Enrichment of glycopeptides by using NH2/ZIC-HILIC MNP 25
2.2.7 Enrichment of phosphopeptides by using NH2/ZIC-HILIC MNP 26
2.2.8 Simultaneous enrichment of glycopeptides and phosphopeptides by pH modulation strategy in peptide mixture 27
2.2.9 Enrichment of glycopeptides from PC9 non-small cell lung cancer cell 28
2.2.10 Simultaneous enrichment of glycopeptides and phosphopeptides by pH modulation strategy from PC9 non-small cell lung cancer cell 29
2.3 Mass spectrometer and data analysis 30
2.3.1 MALDI-TOF MS Analysis 30
2.3.2 Q-TOF LC-MS/MS Analysis 31
2.3.3 Orbitrap LC-MS/MS Analysis 32
2.3.4 Q-TOF LC-MS/MS Data processing and analysis 33
2.3.5 Glycopeptide identification by Byonic software 34
Chapter 3 Results and Discussions 35
3.1 Rationale of Enrichment of Glycopeptides and Phosphopeptides by using Zwitterionic-HILIC (ZIC-HILIC) Magnetic Nanoparticles 35
3.1.1 Magnetic nanoparticle characterization 38
3.2 Method development for single glycoprotein enrichment 39
3.2.1 Enrichment capability for determination of functionalized material on MNP surface 40
3.2.2 Effect of incubation buffer in glycopeptide enrichment by NH2/ZIC HILIC MNP 42
3.2.3 Effect of pH value of incubation buffer on glycopeptide enrichment by NH2/ZIC HILIC MNP 43
3.2.4 Effect of elution buffer on glycopeptide enrichment by NH2/ZIC HILIC MNP 44
3.3 Method development of single phosphopeptide enrichment 45
3.3.1 Effect of pH on phosphopeptide enrichment by NH2/ZIC HILIC MNP 46
3.3.2 Effect of washing buffer on phosphopeptide enrichment by NH2/ZIC HILIC MNP 47
3.3.3 Effect of elution buffer on phosphopeptide enrichment by NH2/ZIC HILIC MNP 48
3.3.4 Optimization of the amount of MNP@ SB/SiO2/NH2 for glycopeptide and phosphopeptide enrichment 48
3.4.1 Simultaneous and sequential enrichment of glycopeptides and phosphopeptides by pH modulation strategy in peptide mixture 49
3.4.2 Simultaneous and sequential enrichment of glycopeptides and phosphopeptides by pH modulation strategy in PC9 non-small cell lung cancer cell 52
Chapter 4 Conclusion 55
References 57
Figures 61
Appendix 77

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指導教授

陳玉如(Yu-Ju Chen)

審核日期

2018-8-22

推文