利用奈米探針結合質譜技術純化與鑑定硫基亞硝基化胜肽

Abstract

硫基亞硝基化為一氧化氮共價結合於蛋白質半胱胺酸硫基之一種可逆的後轉譯修飾，其在細胞內調節訊息傳遞以及生理反應，像是免疫反應、氧化還原調控等。由於S-NO的鍵結不穩定且在生物體中內生性的硫基亞硝基化蛋白質含量低，使得偵測硫基亞硝基化蛋白質以及鑑定修飾位置的方法仍具挑戰與限制。基於Zhang等人的研究，我們使用四種不同的thioester- 以及 ester-phosphine，例如S-propyl 2-(diphenylphosphino) benzothioate (簡稱為L364)以及其餘三種化合物純化硫基亞硝基化蛋白質或胜肽鏈。利用蛋白質酪胺酸磷酸酶1B (PTP1B)其中一已被證實在Cys215會被硫基亞硝基化的胜肽(ESGSLSPEHGPVVVHC215SAGIGR)當作標準胜肽，根據traceless Staudinger ligation-type機制，thioester-phosphine可以直接且專一性的與硫基亞硝基化胜肽鏈的一氧化氮反應並形成雙硫鍵產物。經由LC-MS/MS分析，硫基亞硝基化胜肽鏈的訊號在與這四種化合物反應後有顯著的下降。同時地，可以在Mascot蛋白質資料庫中鑑定到L364與硫基亞硝基化胜肽鏈反應的產物。由於在此四種化合物中L364的反應性較佳，進一步將L364修飾到磁性奈米粒子表面上，以應用於直接且專一性的純化硫基亞硝基化蛋白質或胜肽鏈。此方法應用在化學合成之酪胺酸磷酸酶1B標準胜肽上，並利用基質輔助雷射脫附游離飛行時間質譜儀偵測，其偵測極限可到10 nM；此外，L364@MNP也可將混到牛血清蛋白的酶切胜肽中之硫基亞硝基化蛋白質酪胺酸磷酸酶1B標準胜肽直接且專一性地純化。再者，利用胰蛋白酵素將硫基亞硝基化蛋白質酪胺酸磷酸酶1B水解，也可用L364@MNP直接且專一性地純化出具有硫基亞硝基化的胜肽鏈。此利用磁性奈米粒子結合質譜分析的技術可以提供一個直接且專一性的純化和鑑定硫基亞硝基化胜肽鏈。

S-nitrosylation is a reversible posttranslational modification, covalently interact with nitric oxide (NO) on cysteine thiol group of proteins, involving in a wide range of cellular pathways and physiological processes, such as immune response and redox regulation. Due to the labile nature of S-NO bond and low abundance of endogenously S-nitrosylated proteins in vivo, unambiguous identification of S-nitrosylated proteins and S-nitrosylation sites remains methodologically challenging. Based on the study of Zhang et al., we used 4 different reactive groups of thioester- and ester-phosphine compounds, S-propyl 2-(diphenylphosphino) benzothioate (termed as L364) and others, to enrich S-nitrosylated proteins and peptides. A standard peptide, synthetic ESGSLSPEHGPVVVHC215SAGIGR of protein tyrosine phosphatase 1 B (PTP1B), which has been published for S-nitrosylation in vivo, was S-nitrosylated in vitro. Based on the traceless Staudinger ligation-type mechanism, the disulfide bond was formed when phosphine-ester ligand site-specifically reacted with S-NO bond on S-nitrosylated peptides. LC-MS/MS analysis showed that free S-nitrosylated peptide was significantly decreased after reacting with all ligands, compared to the signal of initial S-nitrosylated PTP1B peptides. Simultaneously, the L364-modified PTP1B peptide was identified by Mascot database search. Due to the better reaction efficiency of L364, the L364 was chose to conjugate on magnetic nanoparticle (L364@MNP) using anhydride linkage for site-specifically enrichment of S-nitrosylated proteins and peptides. Consistent with the appropriate reaction of ligand, S-nitrosylated PTP1B peptide was enriched by L364@MNP. Limit of detection (LOD) experiment showed that about 10 nM of S-nitrosylated PTP1B peptide was enriched and identified by MALDI-TOF. Moreover, the S-nitrosylated PTP1B peptide mixed in tryptic BSA peptide mixture was also site-specifically captured by anhydride-L364@MNP and identified by MALDI-TOF MS. In addition, S-nitrosylated ESGSLSPEHGPVVVHC215SAGIGR from tryptic digested PTP1B protein was also directly and site-specifically enriched by anhydride-L364@MNP. Therefore, anhydride-L364@MNP coupling mass spectrometry analysis can provide a direct and site-specific enrichment for identification of S-nitrosylated proteins and peptides.