親和質譜術應用於特定蛋白質之變異結構檢視與定量的研究

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姓名

莊斯安(Szu-An Chaung) 查詢紙本館藏

畢業系所

化學學系

論文名稱

親和質譜術應用於特定蛋白質之變異結構檢視與定量的研究
(Targeted Protein Quantitation and Variant Screening by Nanoprobe-Based Affinity Mass Spectrometry)

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摘要(中)

疾病的成因常常和蛋白質變異有相當的關聯性，其變異可能來自含量的改變，也可能來自蛋白質結構上的變化，如蛋白質組成胺基酸的缺失(truncation)或胺基酸變異(mutation)。在本篇論文中，我們利用最新發展的磁性奈米結合質譜分析技術(NBAMS)的方法針對疾病相關的標記蛋白質做定性及定量分析。此技術是利用表面裝配抗體的功能性磁性奈米粒子，結合基質輔助雷射游離脫附質譜法(MALDI-TOF MS)，用來對人體血液中的蛋白質做分離和定量的分析以及快速檢視血液中特定蛋白質的同質異構體(protein variant)。
基於抗原與抗體間的作用力，我們挑選了兩個血清中的蛋白質當作為研究的對象，分別是血清澱粉蛋白A(serum amyloid A, SAA)與血清澱粉蛋白P(serum amyloid P, SAP)，利用磁性奈米質譜分析技術可以幫助鑑定人體血清中血清澱粉蛋白A與血清澱粉蛋白P在血清中的同質異構體。本技術最佳化後，我們對正常人與病人血清中這兩種蛋白質之同質異構體所表現出的圖樣做評估；血清澱粉蛋白P與本身的其他兩個醣基化結構在血清中出現的機率並沒有明顯的差異。然而，我們發現血清澱粉蛋白A之同質異構體在這兩個族群具有不一樣的分佈，在眾多包括了缺失以及胺基酸變異的結構之中。我們在胃癌病人中發現了血清澱粉蛋白A中有一個特別的多樣性結構(70%, n=50)是從未被報導過的，而在正常人中沒有觀察到這個多樣性結構(2%, n=50)。有趣的是血清澱粉蛋白A的特別多樣性之結構在食道癌、喉癌，肝癌中也有少量的出現。我們相信這一個這特別的多樣性蛋白質結構與胃癌可能具有高度的相關性，並且與消化系統方面的癌症也有一定關連。此一特別多樣性蛋白質結構的發現是否能作為診斷疾病中有用的指標物或是去探討疾病的成因，仍需要以更大量的檢體去進一步研究。
在傳統基質輔助雷射游離脫附質譜法應用上，定量方法的主要限制來自於非均向之結晶所導致訊號的低再現性。為了克服此限制，我們可藉由外加標準品及提高均勻晶相(seed-layer)的方式，成功的改進了分析物與基質分子在共結晶時的均相性，進而降低了訊號的變異度。經由序列稀釋過後之標準品所建立出的定量曲線可測定及比較正常人與具有心血管疾病病人之血清中的C反應蛋白的含量。同時，我們也證實磁性奈米質譜法所定量出來的結果與酵素免疫分析法所定量出的結果具有一致性。奈米粒子表面上的化學修飾日趨成熟，可針對各式分子裝配作廣泛應用，我們預期本論文發展的磁性奈米質譜法對於疾病機制的研究和臨床上的診斷可有應用的價值。

摘要(英)

Protein variation in disease state not only change in protein content but also varied in protein structure such as protein truncation or mutation. In this thesis, we implemented the newly developed Nanoprobe-Based Affinity Mass Spectrometry (NBAMS) methodology for characterization of disease-related proteins. By a combination of antibody-conjugated nanoprobe and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, the NBAMS is capable of simultaneous enrichment, quantitative analysis and screening of the protein variants in different populations in human plasma.
Based on antibody-antigen interaction, two serum proteins, serum amyloid A (SAA) and serum amyloid P component (SAP) were used as model systems. The NBAMS methodology facilitated multiplexed target protein identification in human plasma, revealing the diverse protein variant of SAA and SAP. After optimization, we evaluated the pattern of multiplexed protein variant between healthy and patient groups. SAP shows no significant difference in its heterogeneous glycosylated forms between these two groups. However, the diverse SAA variants including truncation, mutation and isoforms reveal different pattern in these two populations with statistical significance. A yet-to-be identified protein isoform from the novel allelic variant of SAA was specifically observed in gastric disease (70%, n=50), whereas the isoform was not observed in the normal group (2%, n=50). Interestingly, the novel allelic variants of SAA were also observed in esophagus, liver, and laryngo-carcinoma cancer with lower occurrence. We believe this novel protein variant was highly associated to gastric disease and moderately to the cancers in digest system. Whether the discovery of the novel isoform is correlated with disease mechanism or may serve as a valuable indicator for disease diagnosis remains further investigation with larger population.
The major limitation in quantification by conventional MALDI MS is non-homogeneous crystallization on sample plate that results in poor signal reproducibility. With the assistance of seed-layer surface and spiked internal standard, we successfully reduced signal fluctuation from the improved homogeneous co-crystallization of analyte and matrix molecule. After construction of the calibration curve by a serial dilution of standard protein solution and NBAMS assay, plasma CRP level can be determined in healthy individuals and patients with cardiovascular risk. The quantitative result obtained from NBAMS methodology is consistent to the ELISA measurements. With the flexibility of the functional nanoprobes and diverse application of NBAMS assay, our approach shows great promise in investigating of disease mechanism and clinical diagnosis.

關鍵字(中)

★ 基質輔助脫附游離質譜
★ 生物指標蛋白
★ 血清澱粉蛋白

關鍵字(英)

★ biomarker
★ MALDI-TOF MS
★ serum amyloid a

論文目次

中文摘要……….……………………………………………....………..ⅰ
Abstract…………………………………………………………….....…ⅲ
Table of Contents………………………………………………………..ⅴ
List of Figures and Tables…………….……………………...………….ⅸ
Abbreviations……………………………………………………….......ⅹⅰ
Chapter 1：Introduction….....................................................................1
1-1 Clinical Proteomics and Biomarker…………………...….……………...….1
1-2 Method for Biomarkers Discovery………………………..…………….…..1
1-2.1 2D-PAGE………………………….........................…………………..2
1-2.2 Isotope-coded Affinity Tag combined with
Mass spectrometry………………………………………………..…. 3
1-2.3 Surface-Enhanced Laser Desorption Ionization....……………….…. 4
1-2.4 Post-Translational Modification of Proteins…....………………..…..4
1-3 Method for Biomarker Validation…………………….....................……….5
1-3.1 Enzyme-Linked Immunosorbent Assay (ELISA)….............................5
1-3.2 Protein Array……………………………………….....................……6
1-4 Protein Detection and Quantification by MALDI-TOF……….……..……..6
1-4.1 Limitation of MALDI-TOF in Protein Quantification……….……......….7
1-5Overview of Target Proteins……………………...……………....…….……8
1-5.1 Serum Amyloid A………………………………………….….. .….. 8
1-5.2 C-reactive protein……………………………………...….…….…..9
1-5.3 Serum amyloid P………………………………..…………..….…..10
1-6 Objectives………………………………………………......…….………..10
Chapter 2：Experiment…....................................................................12
2-1 Material………………………….………………………….……12
2-1.1 Chemicals and Materials…….……………………………….12
2-1.2 Synthesis of Antibody-conjugated Magnetic
Nanoparticles….…………………………………….….…12
2-1.3 Human Plasma…………...…………………………..………13
2-2. Mass Spectrometry…….………………………...………………..14
2-3. Method………………………………………………………….14
2-3.1 Immunoaffinity Extraction …………………….……………14
2-3.2 Specificity of Antibody-Conjugated Magnetic Nanoparticles……14
2.3-3 Protein Variant Analysis in Normal and Patient Subject………...15
2-3.4 Seedlayer Surface Preparation……………...……………...…16
2-3.5 Standard Calibration Curve……………...……………………..16
2-3.6 Quantification in Plasma of Different Volume...……………….16
2-3.7 Standard Addition Method for Protein Quantitation……………...17
2-3.8 CRP Quantification in Normal Subje.ct and Patient
with Cardiovascular Risk..………….……….........................17
Chapter 3 Result and Discussion………...…………………………..18
3-1 Workflow for Protein Quantitation and Variant Screening
by NBAMS assay……………………………………………..….18
3-2 Specificity of Antibody-Conjugated Magnetic Nanoparticles …….……19
3-3 Interference form Hemolysis in Human Plasma……………..….…….20
3-4 Selected Protein Enrichment in Human Plasma by NBAMS…………...21
3-5 Protein Variant Screening by Nanoprobe-Based Affinity
Mass Spectrometry (NBAMS)………………………….…..……….22
3-5.1 Optimizations for Protein Variant Analysis……….…….………22
3-5.1.1 Optimization of volume ratio between alpha-SAA and alpha -SAP@MNPs.........................................................23
3-5.1.2 Optimization of Plasma Amount for Multiple Protein
Variants Analysis……………………….….….…….24
3-5.2 Protein Isoform Diversity of Serum Amyloid P and
Serum Amyloid A…………………………………...……..24
3-5.3 Protein Isoform Analysis in Cardiovascular Risk and
Healthy Individuals…………………………………..……26
3-5.4 SAA isoform analysis in gastric cancer, ulcer patient
and normal individuals…………………..……….………...27
3-5.5 SAA isoform analysis in different cancer patients..…..….…….28
3-6.1 Experiment Workflow of Protein Quantification by
NBAMS Assay…………………………….…...…………29
3-6.2 Improved Homogeneity by Pre-crystallization of
Seedlayer Method...………………………………………30
3-6.3 Optimization of NBAMS assay for Protein Quantification….….31
3-6.3.1 Optimization of Spectrum Quality with
Acquisition Method …………………..…………..31
3-6.3.2 Optimization for the Quantity of Internal Standard ...…..32
3-6.4 Comparison of Calibration Curve by conventional and
Modified NBAMS Assay ………………………..………33
3-6.5 Reproducibility of Calibration Curve by the Modified
NBAMS Assay for Protein Quantification ………..…….…..34
3-6.6 Evaluation of Quantification Performance……………...…….35
3-6.7 Quantification of Human Plasma in Patient with Cardiovascular Risk. and Normal Individuals…………...…….36
Chapter 4 Conclusion……………………………....………………..39
Reference............................................................60
Appendix ...........................................................63

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指導教授

陳玉如、丁望賢
(Yu-Ju Chen、Wang-Hsien Ding)

審核日期

2007-7-20

推文