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標題: (1)建立以選擇電子轉移反應偵測模式的胜肽定量新方法(2)利用質譜儀鑑定水稻突變株M0047286榖粒外殼色素成分
(1)New Approaches for Quantitative Peptides Analysis by Selected Electron Transfer Reaction Monitoring (2)Characterization of Pigment in Rice Mutant M0047286 by Mass Spectrometry
作者: 魏碧瑩
Wei, Bi-Ying
關鍵字: mass spectrometry;電子轉移裂解;electron transfer dissociation;peptide quantitative;TNG67;pigment;serotonin;胜肽定量;台農67;色素;血清素
出版社: 分子生物學研究所
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液相層析串聯式質譜儀 (LC/MS/MS) 搭配選擇反應偵測模式 (Selective Reaction Monitoring,SRM) 是質譜分析中常用於胜肽定量的一套策略,此方法能專一針對特定的胜肽片段進行監測。SRM 是先選擇特定質荷比的母離子進行碰撞引致裂解 (Collision-induced dissociation,CID) 後,偵測特定質荷比的子離子片段。低能碰撞引致裂解是目前最常使用的裂解方法,在進行胜肽碎裂的反應會打斷胜肽鍵,利於進行胜肽的序列分析。但胜肽利用碰撞引致裂解方法碎裂之後,母離子會平均碎裂成子離子,因而大幅減低子離子強度。上述的情形容易影響 SRM 的靈敏度。電子轉移裂解 (Electron transfer dissociation,ETD) 為新發展的裂解方法,可補足碰撞引致裂解的缺點,保留蛋白轉譯後修飾的官能基,目前已成為研究蛋白轉譯後修飾的有利工具。然而電子轉移裂解應用在正二價胜肽離子時,經電子轉移後之胜肽離子裂解效率差。在此將電子轉移裂解針對正二價胜肽離子會產生電子轉移而無法有效裂解的缺點加以應用,以發展一套利用電子轉移裂解搭配 SRM 來做為胜肽定量的新方法,命名為選擇電子轉移反應偵測模式 (Selected electron transfer reaction monitoring,SETRM),即選擇特定質荷比的母離子,經電子轉移後偵測電荷還原離子。經實驗證實,針對正二價或正三價的母離子進行定量時,利用 SETRM 的方法能產生比 SRM 更強的離子訊號,因為一般以胰蛋白酶進行水解的胜肽於 LC/MS 分析中,主要為正二價或正三價的母離子為主,因此更適用此套定量方法。其中以 [Glu1]-Fibrinopeptide B Human 標準品測試 SRM 與 SETRM 的定量效果,發現 SETRM 與 SRM 都具有 3 個維度 (order) 的線性範圍,此外因為 SETRM 的定量方法能得到比 SRM 更高的離子訊號,因此期望利用此策略提升質譜儀在胜肽定量上的靈敏度,做為碰撞引致裂解定量時另一互補的方法,提升電子轉移裂解在質譜分析的應用效率。

M0047286為 T-DNA 插入水稻TNG 67所產生的突變株,此突變株具有深褐色穀粒及外殼的外表型。前人研究證實 M0047286 其 286-10 與286-14 基因受到 T-DNA 活化。以酵素活性分析,證實 286-10 與 286-14 皆具有tryptophan decarboxylase (TDC) 的活性,TDC 可將tryptophan 轉換成為 tryptamine。利用基因轉殖技術,獲得能大量表現 286-10 與 286-14 的轉殖株 (Ubi::286-10 與 Ubi::286-14),同樣會產生含有褐色穀粒及外殼的外表型。在本研究中以質譜儀鑑定出在 M0047286 突變株與轉殖株中 serotonin 的含量皆比野生株 (TNG 67) 高。利用試管內進行 serotonin 氧化測試證實,以過氧化氫反應後會產生黃色的色素成分,而經 UV 刺激後則會產生褐色的色素。以質譜分析照射 UV 後變色的 serotonin 標準品,可偵測到 serotonin 形成二聚體的結構。利用液相層析串聯質譜儀分析證實,二聚體的結構在 M0047286 突變株、Ubi::286-10 與 Ubi::286-14的水稻外殼中含量皆高於野生株 (TNG 67)。目前的研究結果無法確定二聚體的結構,但由結果顯示二聚體的含量似乎與水稻外殼的色素變化相關,因此推測 serotonin 二聚體極有可能改變水稻外殼色素。

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) approach with selective reaction monitoring (SRM) allowed for the specific and sensitive quantitation of peptides. SRM is achieved via MS/MS utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions. Low-energy CID tandem mass spectrometry has been, by far, the most common method used to dissociate peptide ions for sequence analysis. However, collisional scattering of product ions in CID results in decrease in intensity of the primary product ion. The lower intensity of the targeted product ion can lead to a reduction in the sensitivity of a quantitative method with SRM. Electron transfer dissociation (ETD) is a prevailing fragmentation method that can be complementary to CID and its utility in sequencing peptides containing post-translational modification. During the ETD reaction, there is a significantly shift toward nondissociative electron transfer as function of decreasing precursor ion charge for doubly charged peptides. In this study, we have developed a method utilizing ETD while monitoring unique precursor to charge reduced ion called selective electron transfer reaction monitoring (SETRM). For +2 and +3 precursor ion, more intense targeted ions for SETRM than SRM that is more suitable to quantify for trypsin digestion peptides. For [Glu1]-Fibrinopeptide B Human, 3 order linearity and excellent accuracy are observed in both SRM and SETRM. Moreover, SETRM provided more signal intensity, expecting to detect lower Limit of detection (LOD) due to a less signal loss compared to SRM analysis. Therefore, a new peptide qualitative and quantitative approach utilizing ETD technique as proteomic tool is provided here.

M0047286 is a rice TNG67 T-DNA insertion mutant showed dark-brown color in leaves, grains and hulls. Genes 286-10 and 286-14 flanked by the T-DNA insertion site is activated in the previous studies. Enzymatic assay using specific substracte by HPLC revealed that 286-10 and 286-14 had tryptophan decarboxylase (TDC) activity, which catalyzes the conversion of tryptophan into tryptamine. Using transgenic approach, transgenic rice Ubi::286-10 and Ubi::286-14 also showed dark-brown hulls. This study LC/MS/MS demonstrated that M0047286 and transgenic rice (Ubi::286-10 and Ubi::286-14) had serotonin compound higher than TNG 67. In vitro oxidation of serotonin by H2O2 and UV radiation results in the eventual formation of yellow and brown pigments, respectively. Mass spectrometry provided evidence for the dimeric serotonin species from UV radiation studies. The serotonin dimer compounds were also detected in M0047286 mutant plant and transgenic rice in LC/MS/MS studies. Some isoforms were detected in the serotonin dimeric sprcies The structure of these isoforms need to be elucidated in the future. In conclusion, the dimeric species of serotonin is strongly related to pigment on rice.
其他識別: U0005-1707200914555900
Appears in Collections:分子生物學研究所

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