親和質譜術應用於蛋白質體學的研究

Affinity Mass Spectrometry for Targeted Proteomics

陳淑華(Shu-Hua Chen)

指導教授 ： 陳長謙

共同指導教授 ： 陳玉如(Yu-Ju Chen)

國立臺灣大學/理學院/化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.10404

摘要

隨著蛋白質體學領域的發展從發現受到調控蛋白質到其功能之驗證，發展高靈敏度及高專一性的標的蛋白質體學的工具日趨重要。在這篇論文當中，我們利用了兩種固態的樣品承載平台(solid supports)：表面經過修飾的聚氟化亞乙烯（PVDF）薄膜以及表面經過設計的奈米粒子（nanoparticle）作為誘餌，發展親和性質譜法。
人體血清是很重要的體液。在病人當中，有某些特定的蛋白質會在血清中大量分泌，在臨床診斷上，這些蛋白質就可以扮演重要的生物標誌（biomarker）。然而，人體的血清當中，蛋白質的含量分佈高達1012之多，是人體衍生出來最複雜的蛋白質體系統，造成了藥物研究及臨床診斷上分析上的極大的困難。在這個研究當中，我們發展了一種簡單有效率的方式，結合了聚氟化亞乙烯輔助免疫親和萃取法(PVDF-aided immunoaffinity assay ) 以及基質輔助雷射脫附游離飛行式質譜儀（MALDI-TOF MS）。基於抗原抗體反應的原理，將抗體固定在PVDF上，我們可以將血清當中的急性期蛋白萃取出來。PVDF 薄膜不僅可以直接當作MALD的樣品盤還可以改善樣品均勻性，增加了以MALDI-TOF MS定量的可行性，利用此法能夠在複雜的樣品當中得到對應的抗原而不需要額外的純化及去鹽。初步結果顯示，PVDF-aided immunoaffinity assay的偵測極限約在nM範圍。另外，在不經過樣品前處理狀況下，我們成功地在血清樣品當中偵測到血清澱粉質P成分（SAP）、血清澱粉質A蛋白（SAA）以及C-反應蛋白（C-reactive）的訊號。非專一性的干擾也在經過選擇適當的阻斷劑（blocking reagents）之後而消除。SAP的定量曲線在10倍的濃度範圍內具有良好的線性關係。我們測得的SAP在正常人血清當中含量平均為41.7± 12.7 mg/L，符合文獻報導的35-40 mg/L。另外我們也發現在胃癌病人當中SAP的含量(39.3±15.8 mg/L)與正常人並沒有明顯的不同，這也與二維膠體電泳（2D gel）的結果具有一致性。
醣的分子辨識在免疫學當中扮演了一個重要的角色。瞭解這種辨識之間的作用可以幫助發展治療感染的新試劑。在這篇論文當中的第二部份，我們發展了一種新的方法，奈米探針輔助親和性質譜法（nanoprobe-based affinity mass spectrometry；NBAMS）。利用表面修飾配基（ligand）的金奈米粒子（AuNP）作為誘餌，可以將所對應的蛋白質在複雜的樣品中同時分離及濃縮，再進一步以MALDI-TOF MS做蛋白質鑑定和配基—蛋白質作用點的定位（epitope mapping）。奈米探針（g-AuNP）是以4 nm 金奈米粒子表面上共價連結半乳糖（galactose）而成，可以由複雜的樣品當中同時的濃縮及分離綠膿桿菌中的親醣蛋白Ⅰ（Pseudomonas aeruginosa lectin I ；PA-IL），並清除其它高含量蛋白質的干擾。可以偵測到約10000倍濃度不同的PA-IL，且偵測極限在nM以下。進一步利用胜肽質量指紋（peptide mass fingerprinting）鑑定，可以100 %序列涵蓋率（sequence coverage）證明了所捕捉到的蛋白質的確為PA-IL。接下來epitope mapping的結果也與文獻當中以X-光繞射所得到結果具有一致性。最後，我們以將包含epitope的胜肽以串聯式飛行質譜法（MALDI TOF/TOF）作de novo sequencing。
我們證明了利用親和性探針搭配質譜技術可以將目標蛋白質作快速且專一性的分離，並可進一步的做定量分析、辨識以及配基—蛋白質作用點的定位。透過簡單有效的PVDF-aided immunoaffinity assay技術，我們可以定量分析具有臨床檢驗價值的蛋白質之含量變化。另一方面，利用NBAMS技術修飾各式各樣配基的奈米探針，可進一步作為尋找與配基有作用力的特定類別分子的工具。

關鍵字

親和質譜術 ； 基質輔助雷射脫附游離質譜術 ； 奈米粒子

並列摘要

As the field of proteomics evolves from the discovery phase to validation and application, tools for targeted proteomics with high sensitivity and specificity remain to be developed. In this study, two solid sample supports, surface modified PVDF and surface engineered nanoparticles were employed as baits to achieve the affinity capture process.
Human serum is an important medium where specific proteins could become elevated in patients and serve as important biomarkers in diagnosis. However, human serum is among the most proteome with a wide dynamic range of more than 1012 in protein abundance. The dynamic range imposes an extreme analytical difficulty for clinical diagnosis. In this thesis, a simple and effective method was developed in combination of PVDF-aided immunoaffinity extraction and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to circumvent the difficulties. Based on antibody-antigen interactions, the acute phase proteins can be selectively isolated from human serum. We show that the polyvinylidene fluoride (PVDF) membrane can be an effective MALDI target probe that improved the spot-to-spot variation for protein detection, allowing the feasibility of quantitative analysis by MALDI-TOF MS. After antibody was immobilized on the solid support, affinity extraction of corresponding antigen can be obtained from complex solution without additional purification and desalting. Preliminary result showed the detection limit of PVDF-aided immunoaffinity assay was in the nanomalar concentrations. To illustrate the efficacy of the approach, serum amyloid P component (SAP), serum amyloid A protein (SAA) and c-reactive protein (CRP) were selectively extracted from human serum with no prior fractionation. Interferences from nonspecific retention were minimized through choice of appropriate blocking reagent on the PVDF membrane. The calibration curve for SAP quantitation revealed at least 1order of magnitude with a good linear response. The measured SAP level in healthy subjects was 41.7± 12.7 mg/L consistent with the literature of 35-40 mg/L. In our experiments, we found that the levels of SAP show no significant change in serum between healthy subjects and patients with gastric cancer (39.3±15.8 mg/L), a result that is in good agreement with the result of 2-D gel experiments.
Molecular recognition events involving carbohydrates play an essential role in various types of interactions in immune system. Knowledge of such interactions can provide new insights in the development of new therapeutic agents for infection treatment. In the second part of this thesis, we report a new approach, nanoprobe-based affinity mass spectrometry (NBAMS), using ligand encapsulated gold nanoparticle (AuNP) as baits for the separation and enrichment of targeted proteins with enhanced detection, protein characterization, and epitope mapping by MALDI-TOF MS. A bioactive and biocompatible nanoprobe was constructed with 4 nm AuNP, which was covalently attached with galactose (g-AuNP). Simultaneous enrichment and isolation of Pseudomonas aeruginosa lectin I (PA-IL) from a mixture without interfere from other high abundance protein was achieved by using g-AuNP. A concentration with 10000-fold difference can be detected successfully and current detection limit was in subnanomoler level. Targeted protein identification by peptide mass fingerprinting with 100% sequence coverage confirmed the identity of captured PA-IL. Subsequent mapping of the epitope containing peptides were consistent with the literature where the binding sites were determined by X-ray crystallographic analysis. Finally, de novo sequencing of the epitope-containing peptide was demonstrated with direct MALDI TOF/TOF.
The combination of biologically active probes with mass spectrometry is capable to rapidly and specifically extracted proteins of interest followed by quantitation, identification and epitope mapping. Through the use of simple and effective purification technique of PVDF-aided immunoaffinity assay, quantitative screening of a clinically protein profiling in a biological fluid can be achieved. The flexibility of versatilely functionalized nanoprobe can be easily adapted for ligand-fishing of class-specific proteins by NBAMS technique.

並列關鍵字

nanoparticle ； MALDI MS ； affinity mass spectrometry

參考文獻

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