開發功能性親和探針結合質譜技術純化硫基亞硝基化胜肽

Development of an affinity probe-based mass spectrometry method for enrichment of S-nitrosylated peptide

林渝沛(Yu-Pei Lin)

指導教授 ： 陳玉如

國立臺灣大學/理學院/化學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10930

摘要

硫基亞硝基化 (S-nitrosylation)為一氧化氮與半胱胺酸形成共價性鍵結的一種可逆後轉譯修飾，在人體之神經、心血管及免疫系統調節訊息傳遞與生理反應。相較於其他後轉譯修飾，硫基亞硝基化形成之共價鍵結較不穩定，且在生物體內的含量較低，因此硫基亞硝基化蛋白質之偵測便成為一大分析挑戰。 生物素轉化技術 (Biotin-switched method)已成為近十年最為廣泛應用在偵測及純化硫基亞硝基化蛋白質之方法，主要藉由蛋白質上的硫基亞硝基化置換為生物素，再利用生物素純化該胜肽或蛋白質，然而生物素轉化技術易產生誤判訊號，繁複的流程造成樣品流失，此技術應用於蛋白質體領域研究仍有待突破，開發一個同時具有準確辨識且快速純化及偵測硫基亞硝基化蛋白質或胜肽的方法為最重要的議題之一。
根據過往所發表的文獻，thioester-phosphine已被指出可直接與亞硝基硫醇進行二次結合反應或一步調控雙硫鍵生成反應。為了開發硫基亞硝基化胜肽或蛋白質之純化方法，我們與國立清華大學林俊成教授合作，合成了兩種具有水溶性的thioester-phosphine化合物，分別為S-(2-(diphenylphosphino)phenyl) butanethioate (簡稱為TEP-L364)及S-ethyl 2-(diphenylphosphino)benzothioate (簡稱為TEP-L350)。其中TEP-L350可以比TEP-L364更穩定存在於複雜生物系統中，且其與硫基亞硝基化胜肽的反應效率較高。經由調整反應酸鹼值及緩衝溶液後，透過LC-MS/MS分析得知，TEP-L350可以得到較高的產物產率。
將TEP-L350與硫基亞硝基化胜肽之反應找出最佳化條件後，便進一步將TEP-L350修飾到磁性奈米粒子以及瓊脂醣珠等固相基材上作為功能性探針。以硫基亞硝基化之酪胺酸磷酸酶1B作為標準胜肽的純化測試結果顯示，透過基質輔助雷射脫附游離飛行時間質譜儀的分析，磁性奈米粒子材料可能有嚴重的非專一性吸附，進而影響探針純化效率；而瓊脂醣珠探針有較好的專一性，其偵測極限可到0.5 nM，在含有15-180倍牛血清蛋白的酶切胜肽混合物或是硫基亞硝基化蛋白質酪胺酸磷酸酶1B之酶切胜肽混合物中，此瓊脂醣珠探針均可以直接純化硫基亞硝基化之酪胺酸磷酸酶1B標準胜肽。此外，藉由N-ethylmaleimide (簡稱NEM)保護(blocking)蛋白質未亞硝化的自由硫醇基，可進一步提升以瓊脂醣珠探針純化硫基亞硝基化胜肽的專一效率。由本論文結果顯示，利用TEP-L350修飾於瓊脂醣珠之功能性探針並結合質譜分析技術，可以提供一個直接且快速純化硫基亞硝基化胜肽鏈之新穎方法。

關鍵字

硫基亞硝基化 ； 功能性親和探針 ； 質譜

並列摘要

S-nitrosylation, reversible covalent attachment of nitric oxide on cysteine residues, involves in cardiovascular, neuron, and immune systems. Due to its labile nature and low abundance, detection of S-nitrosylation is a critical challenge compared to other post-translational modifications. Biotin-switch method is the most commonly used approach for enrichment of S-nitrosylated peptides by replacing the S-nitrosylation sites on proteins with biotin, followed by enrichment with streptavidin-agarose. However, the potential false-positive signals and sample loss during the complicated analytical procedure still limit the applicability of this method on the proteome scale. Therefore, developing a precise and simple method is one of the most important issues for enrichment of S-nitrosylated peptides.
In previously published literatures, the thioester-phosphine has been reported to directly react with S-nitrosothiols by bis-ligation or one-step mediated disulfide bond formation reaction. To achieve enrichment of the S-nitrosylated proteins or peptides, through collaboration with Prof. Chun-Cheng Lin at National Tsing Hua University, we synthesized water-soluble thioester-phosphine ligands, S-(2-(diphenylphosphino)phenyl) butanethioate (termed as TEP-L364) and S-ethyl 2-(diphenylphosphino)benzothioate (termed as TEP-L350). Under presence of cellular nucleophiles, the stability and reaction efficiency of TEP-L350 is higher than TEP-L364. Moreover, TEP-L350 showed good reaction yield. The reaction pH and buffer were optimized for maximum product yield which was examined by LC-MS/MS analysis.
After optimization of the reaction, functionalized affinity probes were fabricated by conjugation of TEP-L350 on the solid supports of either magnetic nanoparticle (TEP-L350@MNP) or agarose resin (TEP-L350@Agarose). For enrichment of standard S-nitrosylated peptides, the magnetic nanoparticle showed serious interference from non-specific adsorption to decrease the enrichment performance. The limit of detection (LoD) of S-nitrosylated standard peptides enriched by TEP-L350-Agarose is about 0.5 nM. The method also showed the successful enrichment and detection of S-nitrosylated standard peptides in mixture containing 15-180-fold non-S-nitrosylated tryptic BSA peptides or tryptic PTP1B peptides. Moreover, NEM (N-ethylmaleimide) blocking of free cysteine residues significantly increased the specificity and enrichment efficiency. Therefore, TEP-L350@Agarose coupling mass spectrometry analysis can be a direct and one-step methodology for enrichment of S-nitrosylated peptides and proteins.

並列關鍵字

S-nitrosylation ； Functionalized affinity probe ； Mass spectrometry

參考文獻

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