本論文的研究目的是發展一套對磺酸化蛋白質萃取和分離的新方法,以利在質譜上分析蛋白質的磺酸化位置和後續的蛋白質體學研究。在氧化還原的訊號機制中可逆半胱胺酸氧化修飾扮演很重要的角色,這篇論文在分析過氧化還原.上半胱胺酸的氧化狀態是一個挑戰。這裡我們提出一個新的方法使用修飾二氧化鈦的奈米鑽石從混和的消化蛋白質中選擇性分離萃取磺酸根胜.包含磺酸和亞磺酸,並經由質譜分析。這個方法不管在低濃度或高複雜的環境下都會選擇性的萃取磺酸化胜.,是一種高效率的萃取工具。實驗室有發表過使用修飾聚精胺酸的奈米鑽石去萃取磺酸化胜.從酵素消化過甲酸氧化的牛血清蛋白上,這裡是補充另外一種濃縮萃取的方法。在MALDI上,我們使用修飾二氧化鈦粒子所萃取到的不同半胱胺酸位置有22個,而在 LC-ESI-MS/MS上我們可以看到所有的半胱胺酸全部35個位置。最後我們應用這個方法搭配LC-MS/MS去辨識過氧化還原.和牛血清蛋白上高度氧化的半胱胺酸位置和數量。在過氧化還原.上的四個氧化半胱胺酸C52, C71, C83, C173,都可以藉由修飾二氧化鈦的奈米鑽石對磺酸的高選擇性萃取。由實驗證明,這個新的萃取方法應用在牛血清蛋白或過氧化還原.上,是可以幫我們了解半胱胺酸的氧化狀態,而進一步去研究探討。
Reversible oxidative cysteine modifications play a critical role in the redox-based signaling mechanism. Dynamic profiling oxidation state of the cysteine residues in protein such as Peroxiredoxin (Prx) presents a formidable challenge. Here, we present a novel approach to selectively isolate sulfopeptides (peptides containing cysteine sulfonic acid and sulfinic acid) from complex digests using TiO2-coated nanodiamonds (TiO2-coated NDs) prior to MS analysis. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a highly complex peptide mixture. This method allowed us to identify the 22 distinct cysteine oxidation status out of a total 35 present in performic-acid-oxidized BSA by MALDI-TOF MS and all the distinct cysteine oxidation status by LC-ESI-MS/MS. Finally, we applied the new approach to identify the cysteine oxidation status of hydrogen peroxide–treated Prx and BSA by LC-ESI-MS/MS. Cysteine residues were found to display in either cysteine sulfonic acid or cysteine sulfinic acid status after hydrogen peroxide treatment. Enhanced detection of low abundance sulfopeptides containing active cysteine positions (C52, C71, C83, and C173) was achieved due to the highly selective enrichment using TiO2-coate NDs.