奈米粒子結合親和質譜術應用於標的蛋白質體學的研究

Abstract

人類的血清是最複雜的蛋白質體之一，含有豐富的物質，其中有一些蛋白質在循環血液中的調控以及表現的量，和人體當時的生理狀態有關，因此血液對於疾病的診斷分析上提供了重要的資訊，而這些帶有資訊的蛋白質也可以更廣泛的定義為生物標記(biomarker)。然而，這些生物標記的含量相當低，遠小於那些在血清中占總蛋白質含量百分之九十九的二十二種蛋白質。要從血液裡偵測到含量低的生物標記時，必須先去除高含量的蛋白質，例如：血清蛋白(human serum albumin)或是免疫球蛋白(immunoglobulin)，這些步驟常造成血液中其他蛋白質的損失。 另一種可供選擇且深具潛力的方式是結合了免疫分析法的概念以及質譜技術的優勢，不僅可以對存在於複雜樣品中的分析物加以分離及偵測，還能鑑定分析物蛋白質的身份。由於磁性奈米粒子(magnetic nanoparticles; mNP)和蛋白質具有類似的尺寸，有利於免疫反應的進行，且很容易利用外加的磁場來簡化整個純化分析的流程，因此可利用磁性奈米粒子簡化目標分析物進行分離和預濃縮的步驟。 在本篇論文裡，可利用表面經過具有高專一性抗體修飾的磁性奈米粒子作為釣餌，用來純化人體血液裡含有和疾病相關的蛋白質，例如:C-反應蛋白(C-reactive protein; CRP)和血清澱粉質P成分(serum amyloid P component; SAP)。整個利用磁性奈米粒子來純化標的蛋白質的實驗過程，最後將結合具有高靈敏度、快速又直接優點的基質輔助雷射脫附游離飛行時間質譜儀(matrix-assisted laser desorption/ionization time-of-flight mass spectrometer; MALDI MS)。實驗結果顯示，靈敏度可以達到subnanomolar (nM)的等級。當進一步將這個方法推廣到複雜的血清樣品時，也就是用來比較正常人和胃癌病人血清中CRP的表現量時，這個方法所測得結果和醫院中常用的酵素結合免疫吸附法(Enzyme-linked immunosorbent assay; ELISA)有非常類似的趨勢。

As one of the most complex proteomes, human serum provides rich informative molecules for diagnostic analyses because the regulation and expression of proteins (putative biomarkers) into the circulating bloodstream are correlated to specific physiological states. Serum contains thousands of proteins, but most abundant twenty-two proteins constitute ~99 % of the protein content. The depletion of high-abundance proteins such as albumin or immunoglobulin is usually an essential yet time-consuming step for low-abundance protein profiling. Alternatively, the combination of immunoassay with mass spectrometry has the potential to detect, isolate, and identify a vast range of target analytes in complex mixtures. Magnetic nanoparticles (mNP) immobilized with antibodies on their surfaces have a promising potential in isolation and preconcentration of targeted proteins. In addition to their comparable dimensions to the protein, the intrinsic magnetic property makes them easy to be manipulated by an external magnetic field for protein purifications. In this thesis, the surface of mNP was covalently encapsulated with specific antibodies which served as “bait molecules” to recognize and isolate disease-related antigens, such as CRP (C-reactive protein) and SAP (Serum amyloid P component) from normal and diseased human sera. The immunoaffinity-captured processes would combine with the power of unambiguous, rapid, sensitive, and “direct” protein identification of MALDI MS. Low abundance proteins in subnanomolar (10-9 ~10-10 M) was successfully detected. When it was applied to screen the CRP and SAP expression profiles of healthy and patient serum suffered from gastric cancer, there was a good consistency between this approach and conventional ELISA readouts.