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標題: | SMA脂質微粒之有效應用於萃取與質譜分析膜蛋白質體及標的膜醣蛋白質 Investigating the use of SMA-lipoparticles in the extraction and mass spectrometry-based analysis of membrane proteome and targeted membrane glycoprotein |
作者: | 王婷 Ting Wang |
指導教授: | 邱繼輝 Kay-Hooi Khoo |
關鍵字: | 人類(醣)膜蛋白,苯乙烯-馬來酸共聚物,原生微環境,醣胜肽,高解析液相析層析串聯質譜, human membrane (glyco)protein,styrene-maleic acid copolymer,native microenvironment,glycopeptide,liquid chromatography with tandem mass spectrometer, |
出版年 : | 2023 |
學位: | 博士 |
摘要: | 膜蛋白占人類基因組的約20%,在各種細胞過程中扮演著重要角色,包括信號、細胞黏附、細胞間通訊和分子運輸。它們對於臨床應用至關重要,例如:成為FDA批准的藥物靶點和診斷生物標記候選物的主要類別。
常規試劑用於溶解膜蛋白及其相互作用者往往具有其不足,可能導致目標蛋白質與脂質雙層的解離,或在高解析液相析層析串聯質譜的分析中引起離子壓制效應,需要進行額外的試劑去除步驟,進而導致樣品損失。苯乙烯-馬來酸共聚物 (Styrene-maleic acid, SMA) 具有將膜蛋白和相關分子封裝在直徑為數十納米的SMA-脂蛋白粒子(SMALPs)中的獨特能力,並可保持蛋白質的原生微環境,包括周圍的脂質和相關分子,從而保留它們的原生結構。這種特性使得可以在不破壞SMALPs下,直接蛋白分解SMALPs中的完整膜蛋白,從而富集醣胜肽,同時減少樣品損失,顯見SMALP在膜醣蛋白研究的潛在優勢。 本論文的研究主要聚焦於探討並優化SMA之使用在膜蛋白質體與膜醣蛋白醣化修飾的有效分析。第一目標是以菌視紫蛋白為模型蛋白,評估不同SMA提取膜的方式取得SMALPs,從而提高提取的蛋白質含量,也同時評估如何降低在蛋白定量上,SMA所造成的干擾,建立一個優化的SMA提取流程並延伸應用於HEK293細胞膜。第二目標旨在建立 SMALP的膜蛋白萃取法之用於約1x10^7至1x10^8有限數量的細胞樣品,進行其膜蛋白質體及交互作用體有效分析的可能性,標靶蛋白為過度表現於HEK293細胞的高度醣化修飾的receptor-type protein tyrosine phosphatase alpha (PTPRA)。雖然實驗得以成功直接對細胞進行SMA溶解後在完整的SMALP上蛋白質水解的膜醣蛋白質分析方法,但經評估後顯示所萃取得的量不足以提供有意義的深度關聯蛋白之鑑定。第三目標則是進一步用所建立且優化的SMALP方法来富集及鑑定PTPRA及其相關蛋白質的醣胜肽。结果顯示,SMA的萃取及直接於SMALP上進行蛋白質水解的流程之用於10^8的細胞數量,較一般常用的 Triton 膜蛋白萃取法可得到更佳的醣胜肽鑑定結果。 整體而言,本研究的重點與主要意義在於首次展示了在有限細胞數量的前提下,直接對所提取得的完整SMALPs進行蛋白質水解,可有效同時進行目標蛋白質體和醣胜肽的鑑定分析。重要的是,所優化的方法在識別醣胜肽的效果超過了傳統常規試劑的方法,突顯了SMALPs 應用於蛋白質體和醣蛋白質體分析研究的潛力,為研究表徵蛋白質修飾和相互作用開啟了新的途徑。 Approximately 20% of the human genome encodes for membrane proteins, which play fundamental roles in various cellular processes, including signal transduction, cell adhesion, cell-cell communication, and molecular transport. They are crucial for clinical applications, such as being the main category of FDA-approved drug targets and diagnostic biomarker candidates. Conventional reagents used to solubilize membrane proteins and their interactors would dissociate target proteins from lipid bilayers and induce ion suppression effects during liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. The latter limitation requires an additional detergent removal step, often resulting in sample loss. Styrene-maleic acid copolymers (SMA) possess a unique capability to encapsulate transmembrane proteins and associated molecules within SMA-lipoparticles (SMALPs) that have a diameter of tens of nanometers. It will maintain the native microenvironment of the enclosed proteins, including the surrounding lipids and associated molecules, thereby preserving their native structures. In analyzing membrane glycoproteins, these characteristics of SMALPs potentially allow for direct digestion and enrichment of glycopeptides from SMALP-encapsulated intact transmembrane proteins without first extracting them out of the membrane. It may thus reduce sample loss and facilitate subsequent glycopeptide analysis. In this thesis work, how SMA may be utilized to enable a more efficient and productive membrane proteomics and protein glycosylation analysis was systematically investigated and optimized. The first objective was to evaluate different methods to produce SMALPs from membrane fractions and enhance the proteomic content derived thereof, using bacteriorhodopsin as the model target protein. The technical challenges arising from SMA interference in protein quantification were also addressed and the developed workflow was subsequently applied to HEK293 cell membranes. The second objective involved establishing the feasibility of carrying out SMALP-based membrane proteomic and possibly interactomic analysis using a limited quantity of starting biological materials as presented by 1x10^7 to 1x10^8 HEK293 cells overexpressing a heavily glycosylated target membrane glycoprotein, receptor-type protein tyrosine phosphatase alpha (PTPRA). Successful proteomic analysis was achieved by directly proteolyzing intact SMALPs derived from intact cells but, despite confidently identifying a handful of co-isolated proteins, the yield was insufficient for a more meaningful, in-depth analysis of the proteins associated and interacting with PTPRA. The third objective focuses on utilizing the SMA-based method to enrich glycopeptides from PTPRA and its co-localizers for subsequent LC-MS/MS-based identification. Compared to conventional Triton-based extraction, it was found that the SMALP-based method consistently afforded more efficient glycopeptide identification, when the starting amount of cells was limited. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89929 |
DOI: | 10.6342/NTU202303159 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 生化科學研究所 |
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