(2) 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。 (1) 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.
(2) 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.