以金屬親和層析法結合質譜技術分析磷酸化蛋白體

Qualitative and Quantitative IMAC-based Strategy Development toward Comprehensive Phosphoproteomics Analysis

蔡家烽(Chia-Feng Tsai)

指導教授 ： 陳玉如

國立臺灣大學/理學院/化學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.10111

摘要

蛋白質磷酸化在細胞訊息傳遞及功能調控上扮演關鍵角色。然而磷酸化含量的改變往往驅動與活化整個系統下游的訊息傳導路徑。過去幾年，磷酸化蛋白質體學技術已日趨成熟。但現今定量策略仍然無法有效區分所偵測到磷酸化的變化是來自於蛋白質含量的改變抑或是磷酸化程度上的改變。
在本論文第一部分中，我們首先發展一金屬導向序列純化金屬親和層析法來有效分離不同種類的磷酸化胜肽。此方法利用鎵金屬與磷酸根的親和能力相對較弱，可將高達99.4%的具有更多負電荷的多磷酸化胜肽純化出來。此外，藉由與磷酸化官能基親和能力不同的鎵與鐵兩種金屬離子，可有效地將酸性 (72%)、鹼性 (85%) 以及帶有脯氨酸(Proline)(79%) 的這些不同蛋白激酶受質的酶磷酸化胜肽分別在第一維鎵金屬以及第二維的鐵金屬親和層析法分離出來，利用此方法有效的降低樣品複雜度以提升鑑定到與肺癌相關訊息傳導路徑蛋白的數量。
在論文第二部分中，為了能夠分析單一細胞的磷酸化程度，我們發展一以特定序列標靶導向的蛋白質體定量平台。至今，此平台為第一個可用來系統性的針對單一人類蛋白質體進行磷酸化程度定量分析。藉由蛋白激酶與其受質的反應專一性，從僅僅50微克的細胞量中，我們可定量到上百條低含量的酪胺酸的磷酸化胜肽。利用此特定序列標靶導向的蛋白質體定量平台，我們找到幾個蛋白，其磷酸化程度的改變可能與肺癌抗藥機制有關。此外，透過建立EGFR和CK2兩個蛋白激酶的訊息網路，我們可以從中找出具有潛力的肺癌抗藥標靶蛋白。
在論文最後一部分中，我們發展了一高解析度的掃描頻率多重反應監測質譜法(Enhanced Duty Cycle Multiple Reaction Monitoring,EDC-MRMHR) ，可以進一步驗證膜蛋白以及磷酸化位點進行準確定量分析。藉由增強在飛行式質譜其掃描頻率週期 (Enhanced Duty Cycle) ，針對碎片離子偵測靈敏度提高了10倍。此定量方法提供了好的定量精準度以及線性範圍(R2 =0.998, CV<20%)。我們利用此方法於10位大腸直腸癌病患中驗證潛在的癌症生物標記。其結果與傳統利用抗體驗證的方法一致。我們期待此論文中所發展出新的分析策略在將來可有效地應用在不同疾病的磷酸化蛋白質體學的分析。

關鍵字

磷酸化蛋白體 ； 金屬親和層析法 ； 磷酸化程度 ； 高解析度的掃描頻率多重反應監測質譜法 ； 肺癌 ； 大腸癌

並列摘要

Measurement on the phosphorylation event is often used as indicator for signaling pathway activation. Despite that analytical approaches for phosphoproteomics have matured within the past decade, enrichment and detection towards comprehensive phosphoproteomic profiling remain to the improvement. In addition, the functional role of aberrant protein phosphorylation in cancer-related cell signaling is impacted by its fractional stoichiometry. However, the interpretation of quantitative phosphoproteomics studies by conventional approaches is complicated because each differential phosphorylation event can be associated with a change either in phosphorylation stoichiometry or in protein abundance. Most of the current quantitative strategies are not able to differentiate such changes.
In this thesis, we developed multiple approaches to address the above issues. To overcome the complexity of the human phosphoproteome, in the first part of thesis, we introduced a tip based immobilized metal ion affinity chromatography (Ga3+-Fe3+-IMAC) for sequential enrichment of heterogeneous phosphopeptides. Based on the weak binding affinity between Ga3+ and phosphopeptides, the Ga3+-IMAC successfully enhanced the enrichment of multiple phosphopeptides, which carried more negative charges; 99.4% were enriched by the first Ga3+-IMAC. Acidophilic phosphorylation sites were predominately enriched in the first Ga3+-IMAC (72%), while Pro-directed (85%) and basophilic (79%) phosphorylation sites were enriched in the second Fe3+-IMAC. This strategy provided complementary mapping of different kinase substrates in multiple cellular pathways related to cancer invasion and metastasis of lung cancer.
To measure the phosphorylation stoichiometry of single cell status, in the second part of the thesis, we have developed a motif-targeting quantitative proteomic approach. To our knowledge, this method is the first large-scale approach to provide system-wide phosphorylation stoichiometry of a single-state human proteome. In addition, this assay offers the advantage of kinase-targeted complexity reduction for deeper phosphoproteome analysis; hundreds of tyrosine phosphopeptide could be detected from only 50 μg lysates. For the application of quantitative measurement on the drug-resistant and sensitive lung cancer cell line, this motif-targeting approach differentiates drug resistance-associated changes in phosphorylation stoichiometry from those at the protein level in lung cancer, which also suggested potential druggable target protein in the EGFR- and CK2-centred kinase-substrate network.
In the last part of thesis, we developed a multiple reaction monitoring (MRM) based quantitative platform, EDC-MRMHR, as an alternative method for verification of site-specific alteration of phosphoprotein, especially for antibody

並列關鍵字

phosphoproteomics ； IMAC ； phosphorylation stoichiometry ； EDC-MRMHR ； lung cancer ； colon cancer

參考文獻

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

2. Rane, S. G., and Reddy, E. P. (2000) Janus kinases: components of multiple signaling pathways. Oncogene 19, 5662-5679

3. Giamas, G., Stebbing, J., Vorgias, C. E., and Knippschild, U. (2007) Protein kinases as targets for cancer treatment. Pharmacogenomics 8, 1005-1016

4. Daub, H. (2010) Kinase inhibitors: narrowing down the real targets. Nat. Chem. Biol. 6, 249-250

5. Malumbres, M., and Barbacid, M. (2007) Cell cycle kinases in cancer. Curr. Opin.