開發醣基化及磷酸化修飾膜蛋白體之 同步定量分析策略

Development of Quantitative Strategy for Simultaneous Analysis of N-glycosylated and Phosphorylated Membrane Proteome

簡芷薇

指導教授 ： 林俊成 ； 陳玉如

國立清華大學/理學院/化學系/博士(2013年)

摘要

細胞膜蛋白質的活性主要受其轉譯後修飾所調控，如位於細胞膜外區域之醣基化修飾扮演著訊息接收的功能，而細胞膜內區域之磷酸化修飾則開啟下游的訊息傳遞路徑。然而，醣基化與磷酸化修飾的膜蛋白體在性質上因具有高疏水性、低化學劑量及質譜分析中較不易游離化而導致的離子抑制效應，和其同時具有相異的修飾型態使得其在蛋白體分析上仍然是一個相當大的挑戰。因此，本研究目的為開發一新穎分析策略，以去醣基化為基礎的圖譜標的法並結合固化金屬親和層析法(IMAC)與同位素標記相對與絕對定量(iTRAQ Labeling)之策略，以期能同時定量分析細胞膜上之醣基化蛋白質體與磷酸化蛋白質體及其修飾位置。
本研究藉由去醣基化胜肽(deglycopeptide)其游離效率較強而易出現於質譜圖中，來幫助鑑定醣基化胜肽(glycopeptide)。此概念首先利用HeLa細胞株來證明其可行性。圖譜依據兩項準則進行篩選:1) 皆具有共同序列(consensus sequence) Asn-Xxx-Ser/Thr而造成胜肽分子量1Da的位移；2) 胜肽去醣基化後，所得訊號強度比例達二倍的增加。值得注意的是，去醣基化後的膜蛋白樣品與原本樣品相比，其胜肽產量約有1.25倍的增加。再者，此分析方法最重要的優勢在於能同時鑑定和定量醣基化胜肽/磷酸化胜肽修飾及他們相對應的蛋白質在不同生物狀態下的表現量。因此，我們進一步將此分析分法結合了固化金屬親和層析法並應用在分析受到細胞白介素第二、五因子(interleukin 2/5)刺激之後的B細胞(BCL-1)其膜蛋白之醣基化與磷酸化修飾的改變。此外，在利用酵素(N-Glycosidase F, PNGase F) 進行去醣基化反應中同時加入了同位素18O標記於胺基酸Asn上，使得醣基化胜肽的鑑定更加可信。而在定量分析上，表現量上差異的醣基化蛋白質與磷酸化蛋白質顯示了許多蛋白質的醣基化與磷酸化參與了B淋巴癌細胞株中的免疫反應及細胞存活等訊息路徑。本研究期望能針對醣基化及磷酸化修飾之膜蛋白體的同步定量分析提供一個新穎的分析策略。
第二部分，本研究利用凝集素表面修飾上硼酸官能基所開發新混合型材料，BAD-lectin，應用於細胞層次的醣蛋白體分析。研究中使用了三種BAD-Lectin@MNPs,，其在醣基化胜肽純化上顯示了良好的專一性。再者，利用不同的質譜儀分析方法，三種BAD-Lectin@MNPs所純化出來之醣基化胜肽皆俱有其不同凝集素之相對應的特性醣鏈分子。我們期望此一新型材料結合質譜分析法能提供為偵測醣分子探針之工具，以利更深入的醣蛋白體分析。

關鍵字

膜蛋白質體學 ； 醣基化 ； 磷酸化 ； 質譜分析

並列摘要

The activity of membrane proteins are critically controlled by post-translational modification (PTM), such as glycosylation in the extracellular domain that functions as signal receiver, while the phosphorylation occurring at the intracellular domain initiates the signaling transduction pathway. However, it is still a great challenge to analyze them due to the bottlenecks of highly hydrophobic nature of membrane proteins, low stoichiometry on modification, heterogeneous PTM pattern and ion suppression effect from unmodified peptides of high abundance. To facilitate concomitant analysis of membrane proteome and their PTM sites, we proposed a quantitation platform by integration of deglycosylation-based spectra enrichment, IMAC enrichment and iTRAQ labeling strategies. The performance of spectra enrichment was first demonstrated the glycopeptides can be identified based on two criteria: 1) consensus sequence Asn-Xxx-Ser/Thr with mass shift of 1 Da; 2) iTRAQ ratio with 2-fold enhancement. Further incorporation of 18O-labelig in the sequential digestion steps, the iTRAQ-based signal enhancement with 18O-labeled deglycosylation achieved 100% confidence for accessing glycosylation site occupancies. This strategy also offers advantage to discriminate alterations at either the protein expression level or the modification extent on glycosylation and phosphorylation under different biological states. We integrated this strategy with IMAC purification for quantitative analysis on the B cell lymphoma in response to cytokine stimulation. The differential expression of glycosylation sites and phosphorylation sites revealed that many proteins involved in the immune response and receptor signaling pathway from B cell lymphoma in response to IL-2/IL5 stimulation. We expected that this spectra enrichment strategy may provide a new method for concomitant analysis of N-glycosylated and phosphorylated membrane proteome.
Next, a new type of hybrid biomaterial, namely a boronicacid-decoratedlectin (BAD-lectin), for efficient bifunctional glycoprotein labeling and enrichment was developed and applied on the level of whole cell lysates for glycoproteomic analysis. Three different types of BAD-lectin@MNPs exhibited excellent specificities for glycopeptide enrichment. Furthermore, the precursor ion discovery (PID) mode from Q-TOF-MS was used to filter glycopeptide spectra, the enrichment selectivity was observed that each BAD-ConA@MNP, BAD-AAL@MNP and BAD-SNA@MNP contain the majority of characteristic fragments of their corresponding recognition glycan. With the demonstrated enrichment selectivity and enhanced extraction efficiency, the reported BA-Lectin@MNP-based mass spectrometric method provides a glycan-targeting tool to facilitate the in-depth analysis of glycoproteome.

參考文獻

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