結合化學修飾與質譜分析方法鑑定受特定去乙醯酶作用之乙醯化受質蛋白體
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2017
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離胺酸上的乙醯化(acetylation)是一種可逆的蛋白質後轉譯修飾，藉由乙醯基酶(lysine acetylase)加上乙醯基和去乙醯酶(lysine deacetylases)移除乙醯基所調控。蛋白質上的乙醯化在基因的轉譯上扮演著很重要的角色，包括去氧核醣核酸和蛋白質間的相互作用，蛋白質的穩定性，蛋白質的位置和轉譯的活性等。因此，了解細胞中不同去乙醯酶所特別調控的受質蛋白及修飾位點尤其重要。
我們開發了一個利用化學修飾的方法，分別選用SIRT1和HDAC1兩種去乙醯酶作為此方法開發的模型，此方法結合化學標定以保護離胺酸之一級胺反應、去乙醯基反應，並將去乙醯的位點接上生物素，進而利用生物素和抗生物素蛋白的高親和作用力進行專一性純化，再利用質譜分析以鑑定去乙醯酶之受質蛋白及乙醯化的特定位點。為了驗證新方法的每一實驗步驟，利用兩段合成的胜肽，H3K4(SIRT1受質)和H3K27(HDAC1受質)進行每一步驟的確認，並優化每步驟的條件。再者，為了驗證此方法在複雜樣品的可行性，分別將H3K4和H3K27胜肽混進BSA蛋白質中進行驗證和鑑定，成功地在質譜中鑑定到H3K4和H3K27這兩條胜肽及其修飾位點，結果顯示純化H3K27的專一性為63.8%。我們將此方法應用於癌細胞(Hela cell)，希望能分析被特定去乙醯酶作用受體。為了增加鑑定到受質位點的機會，我們改良第一步驟化學標定以保護離胺酸的副產物，利用磺基-N-羥基琥珀酰亞胺-乙酸酯(sulfo-NHS-acetate)作為保護離胺酸的試劑，經過了條件優化後，成功地鑑定到H3K4和H3K27兩段胜肽並鑑定到812個被HDAC1去乙醯酶作用受質蛋白，其中包含1870條受質胜肽，其中40條受質胜肽為前人研究已經報導的受質蛋白。目前，我們尚未成功地鑑定到被SIRT1去乙醯酶作用受質胜肽，希望未來能找出原因並且解決問題。
Lysine acetylation is a reversible posttranslational modification (PTM) of proteins regulated by lysine acetylase (KATs) and lysine deacetylases (KDACs). Protein acetylation plays a key role in regulation of gene expression involves in many biological process, such as protein interaction, activity, and localization. The knowledge for substrate specificity of KDACs is essential for understanding the role of an individual KDAC in a particular cellular process. In this study, we attempted to develop a chemical modification method, combining lysine blocking, deacetylation, and tagging by biotylation, combined with mass spectrometry analysis for delineating deacetylase-specific acerylproteome. Two synthetic standard peptides H3K4 (SIRT1 substrate) and H3K27 (HDAC1 substrate) was used to verify our workflow. First, the N-terminal amino acids and unmodified lysines (ε-nitrogen of lysine) will be chemically derivatized by adding propionic anhydride. Substrate sites were deacetylated by specific deacetylase, SIRT1 and HDAC1. Here, the reaction time and temperature for deacetylation reaction were optimized. Deacetylated lysine was biotinylated by sulfo-NHS-S-S-Biotin for enrichment of labeled peptides by strepavidin beads. To release the peptides from beads, reduction and alkylation reaction were performed by adding DTT and IAM. The identification of the modified sites was achieved by tandem mass spectrometry. In the preliminary result, the feasibility of this method was demonstrated by identification of H3K4 and H3K27, which were spiked into BSA and the specificity of enrichment efficiency is 63.8%. For reducing side-products which produced from propionylation, we substituted propionic anhydride to sulfo-NHS-acetate for lysine blocking and successfully verified new workflow by spiking H3K4 and H3K27 for identification. We identified 812 substrate proteins including 1870 HDAC1 substrate peptides and 40 substrate-peptides from known substrate proteins in literature. However, we did not identify any SIRT1 substrate proteins yet, in the future, we will further modified the identified substrate and their acetylation sites.
Lysine acetylation is a reversible posttranslational modification (PTM) of proteins regulated by lysine acetylase (KATs) and lysine deacetylases (KDACs). Protein acetylation plays a key role in regulation of gene expression involves in many biological process, such as protein interaction, activity, and localization. The knowledge for substrate specificity of KDACs is essential for understanding the role of an individual KDAC in a particular cellular process. In this study, we attempted to develop a chemical modification method, combining lysine blocking, deacetylation, and tagging by biotylation, combined with mass spectrometry analysis for delineating deacetylase-specific acerylproteome. Two synthetic standard peptides H3K4 (SIRT1 substrate) and H3K27 (HDAC1 substrate) was used to verify our workflow. First, the N-terminal amino acids and unmodified lysines (ε-nitrogen of lysine) will be chemically derivatized by adding propionic anhydride. Substrate sites were deacetylated by specific deacetylase, SIRT1 and HDAC1. Here, the reaction time and temperature for deacetylation reaction were optimized. Deacetylated lysine was biotinylated by sulfo-NHS-S-S-Biotin for enrichment of labeled peptides by strepavidin beads. To release the peptides from beads, reduction and alkylation reaction were performed by adding DTT and IAM. The identification of the modified sites was achieved by tandem mass spectrometry. In the preliminary result, the feasibility of this method was demonstrated by identification of H3K4 and H3K27, which were spiked into BSA and the specificity of enrichment efficiency is 63.8%. For reducing side-products which produced from propionylation, we substituted propionic anhydride to sulfo-NHS-acetate for lysine blocking and successfully verified new workflow by spiking H3K4 and H3K27 for identification. We identified 812 substrate proteins including 1870 HDAC1 substrate peptides and 40 substrate-peptides from known substrate proteins in literature. However, we did not identify any SIRT1 substrate proteins yet, in the future, we will further modified the identified substrate and their acetylation sites.
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Keywords
化學修飾, 質譜, 去乙醯酶, 乙醯化受質蛋白體, Lysine acetylation, SIRT1, HDAC1, Acetylproteome, Deacetylase-specific acerylproteome