開發免標定定量法結合質譜技術之硫基亞硝基化蛋白質體研究

Label-free strategy for site-specific quantitation of S-nitrosylome

周曉喬(Hsiao-Chiao Chou)

指導教授 ： 陳玉如

國立臺灣大學/理學院/化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10661

摘要

蛋白質硫基亞硝基化是一類經由一氧化氮調控的新興蛋白質轉譯後修飾，此修飾可專一性地作用在特定的半胱氨酸殘基上，並藉此在許多生物功能及訊息傳遞上扮演著關鍵的調節角色。儘管此調控具有重要性，由於硫基亞硝基 (S-NO) 鍵結的不穩定及低含量的內生性硫基亞硝基化蛋白質，導致在研究蛋白質硫基亞硝基化的方法上面臨許多挑戰。因此本論文的第一個部分，是建構改良式類生物素置換法策略，以對硫基亞硝基化之蛋白質及其修飾位置具有位點特異性之辨識。透過使用硫基烷基化反應試劑，碘乙酰胺及PEO-biotin，的改良式類生物素置換法並結合串聯式質譜檢測，此法提出一個直接並明確對硫基亞硝基化位置具有選擇性辨別的分析策略。
於論文的第二部分，則是發展一個結合改良式類生物素置換法策略之免標定定量法，並在搭配液相層析串聯質譜儀下，用以位點特異性之辨識及定量生物體內之蛋白質硫基亞硝基化。為了增加定量的準確度，經由硫基亞硝基化的內標蛋白質會被加入到欲進行改良式生物素置換法的分析樣品蛋白質中，使其一同進行改良式生物素置換法、酵素水解、純化，以及液相層析串聯質譜儀分析。在外加不同含量之硫基亞硝基化卵白蛋白至經由 S-nitroso-N-acetylpenicillamine (SNAP) 和左旋半胱氨酸刺激之內皮細胞溶包產物內，可建立出一條在四百倍變化範圍內具有良好的線性 (R2 = 0.987) 的定量曲線。透過二重複實驗之比較，其結果證實此法為高精準度和低誤差值 (兩倍標準偏差值為 0.2)，進而顯示此定量平台可以穩定及有效的用以辨識及定量生物體內之蛋白質硫基亞硝基化。而在刺激後，共有 308 個帶有過量表現硫基亞硝基化修飾的蛋白質可被定量於二重覆實驗中。其中有些可定量之硫基亞硝基化蛋白質被發現參與許多和一氧化氮相關的生物路徑，像是抗氧化訊息傳遞。更重要的是當中有些硫基亞硝基化作用位置未知之硫基亞硝基化蛋白質，其上可被硫基亞硝基化之半胱氨酸殘基已被本方法無偏差的報導出來，像是蛋白質降解脢等。因此，此數據證實結合改良式類生物素置換法策略之免標定定量法是一有效的蛋白質體策略可同時用於位點特異性的辨識硫基亞硝基化蛋白質及定量硫基亞硝基化蛋白質體之動態變化。據我們所知此法是目前第一個成功用免標定定量法之蛋白質體學研究策略用以探討蛋白質硫基亞硝基化。

關鍵字

硫基亞硝基化 ； 類生物素置換法 ； 免標定 ； 定量蛋白質體學 ； 氧還反應調節

並列摘要

S-nitrosylation, an emerging post-translational modification (PTM) specifically on cysteine residues by nitric oxide (NO), plays important roles to regulate cellular functions and signaling events. Despite its importance, the study of S-nitrosylation remains methodologically challenges because of the lability of S-NO bond and low-abundant level of endogenously S-nitrosylated proteins. In the first part of thesis, a modified biotin switch method was presented to site-specifically identify S-nitrosylated protein and modification site. Using S-alkylating agents, IAM and PEO-biotin, in modified biotin switch method coupled with tandem mass spectrometric analysis, this protocol provided a direct site-specific identification method, allowing unambiguous differentiation of the S-nitrosylated cysteine site.
In the second part of thesis, a label-free quantitative approach combining modified biotin switch method and LC-MS/MS analysis was developed for site-specific identification and quantitation of in-vivo S-nitrosylation. For accurate quantitation, S-nitrosylated internal standard proteins were spiked into sample proteins and subjected to modified biotin switch method, digestion, purification, and LC-MS/MS analysis. A reliable linear calibration curve with 400-fold dynamic range (R2=0.9873) for quantitation was established by spiking different amount of S-nitrosylated ovalbumin into SNAP/L-cysteine-treated endothelial cell lysate. The results obtained from duplicate experiments revealed high accuracy and low variation (2 SD = 0.2), demonstrated that this strategy offer a stable and efficient platform for identification and quantitation of S-nitrosylation. The preliminary quantitative results revealed that 308 proteins with over-expressed S-nitrosylation signals were quantified in replicate experiments after stimulation. Some of these proteins have been found to be involved in NO-related processes, such as oxidative stress signaling pathway. Most importantly, the unknown sites of some previously reported S-nitrosylated targets were site-specifically identified by our approach, such as proteasome, etc. Therefore, the date demonstrated that the label-free quantitative approach combining modified biotin switch method was an effective proteomic approach for site-specific identification of the S-nitrosylated targets as well as quantification of the dynamic change of S-nitrosylation. To our knowledge, this study was the first quantitative proteomic for protein S-nitrosylation by label-free strategy.

並列關鍵字

S-nitrosylation ； Biotin switch method ； Label-free ； Quantitative Proteomics ； redox modulation

參考文獻

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