開發奈米探針結合質譜技術之磷酸蛋白質體研究

儘管調控蛋白質磷酸化修飾在細胞訊息傳遞的過程中扮演了重要的角色，針對單一以及多重磷酸化胜肽進行全面性的定性以及定量分析仍面臨嚴峻的挑戰。在定性分析方面，奈米粒子已經廣泛的被使用在做為特定生物分子探針。在本篇論文中，開發了表面包覆聚乙二醇之奈米探針金屬親和層析法(Surface Blocked Nanoprobe-Based Immobilized Metal Affinity Chromatography, NB-IMAC)進行全面性的純化單一以及多重磷酸化的胜肽。此方法可以從高度稀釋的酪蛋白中同時純化出單一以及多重磷酸化的胜肽。藉由聚乙二醇包覆奈米粒子表面，調控系統溶液醋酸的濃度，反應溶液的pH值以及奈米粒子對蛋白質的重量比例，我們從四百毫克的Raji B cell中純化並鑑定出1283條磷酸化胜肽，專一性達百分之八十。與毫米等級的金屬親和層析法比較，奈米金屬親和層析法鑑定到兩倍數目的多重磷酸化胜肽。在定量分析方面，我們結合了免標定四維校正演算法以及表面包覆聚乙二醇之奈米粒子金屬親和層析法探討人類胚胎幹細胞分化過程中蛋白質磷酸化的變化。此方法可以鑑定且定量402條磷酸化胜肽(218個磷酸化蛋白)。從初步的資料分析中，發現在人類胚胎幹細胞分化的過程中蛋白質磷酸化有劇烈的改變，並且與已發表的文獻比對發現藉由磷酸化特定位置以活化胞外訊號調節激脢(ERK)是人類胚胎幹細胞分化的重要指標。因此，表面包覆聚乙二醇之奈米粒子金屬親和層析法提供一個具有高靈敏高專一性以及有效率的定性以及定量分析磷酸化蛋白質體學的研究平台。

關鍵字

磁性奈米粒子；金屬親合層析法；質譜；磷酸蛋白質體學；免標定定量法；人類胚胎幹細胞

並列摘要

Despite the significant regulatory role of protein phosphorylation in cellular signaling, comprehensive characterization and quantitation of site-specific phosphorylation still remained challenges, especially for multiply phosphorylated peptides. Due to the unique features of surface area-to-volume ratio, magnetic separation and flexible surface functionalization, magnetic nanoparticles have been widely fabricated as biological probes. Here we demonstrate a surface-blocked nanoprobe-based immobilized metal affinity chromatography (NB-IMAC) for high performance phosphopeptide enrichment. This approach show unbiased extraction of both mono- and multiply phosphorylated peptides from highly diluted β-casein solution at 2×10-10 M. By surface blocking with polyethylene glycol, controlling the concentration of acetic acid and pH value, and adjusting MNP-to-protein ratio, 1283 phosphopeptides (false discovery rate is 2.9%) could be identified 400 microgram Raji B cell with nearly 80% purification specificity. Notably, two-fold increment of multiply phophorylated peptides was achieved by NB-IMAC compared to the micro-scale IMAC. For further quantitative phosphoproteomics analysis, we integrated the NB-IMAC protocol with LC-MS/MS to develop a label-free quantitation method. Using replicate cell lysate from human embryonic stem cells, the quantitation strategy provides narrow distribution of log2 ratio -0.05 (2SD = 95% confidence), in consistent with expected peptide ratio of 1. Human embryonic stem cells (hESCs) have generated an enormous interest because of its pluripotent ability to differentiate into various types of cells. We applied NB-IMAC protocol to quantitatively analyze the differentiation-mediated phosphoproteomic alteration during embryonic differentiation. After cell proteolysis, phosphorylated internal standard was spiked into the undifferentiated hESCs or differentiated hESCs and subjected to gel-assisted digestion, NBIMAC purification and LC-MS/MS analysis. Without any fractionation procedure, 402 phosphopeptides and 203 phosphoproteins were quantified. The preliminary data revealed 132 up-regulated and 245 down-regulated phosphorylated sites, indicating dramatic change in phosphoproteome during the process of embryonic differentiation. Among these phosphorylation sites, activated ERK 1/2 by phosphorylation at Thr 202 and Thr 184 site in differentiated state of embryoid bodies (EBs) has been previously reported to play an important role in cell differentiation. We expect that this newly developed phosphopeptides purification and quantitation strategy will offer a generic platform to investigate differential phosphorylation levels.

並列關鍵字

Magnetic Nanoparticle ； Immobilized metal ion affinity chromatography ； Mass spectrometry ； Phosphoproteomics ； Label-free Quantitative method ； Human embryonic stem cell

參考文獻

1. Hunter, T., Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling. Cell 1995, 80, (2), 225-236.

2. Lee, M.; Goodbourn, S., Signaling from the cell surface to the nucleus. Essays Biochem. 2001, 37, 71-85.

3. Hunter, T., Signaling--2000 and Beyond. Cell 2000, 100, (1), 113-127.

5. Johnson, L. N.; Lewis, R. J., Structural Basis for Control by Phosphorylation. Chem. Rev. 2001, 101, (8), 2209-2242.

6. Lois, E. R.; Shelley, J. R.; David, H. F., From ion currents to genomic analysis: Recent advances in GABAA receptor research. Synapse 1995, 21, (3), 189-274.

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開發奈米探針結合質譜技術之磷酸蛋白質體研究

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