In Vitro Stable Isotope Labeling For Metabolite Analysis Of Plant

Electrospray ionization (ESI) is a soft ionization technique, which can ionize and transfer the analytes directly from liquid solution into gas phase without destroying the structure of the analytes.The invention of ESI largely promote the development of coupling technique of liquid chromatography and mass spectrometry (LC-MS). Today, LC-MS has been widely used in quantitative and qualitative determination of large biomolecules, organic small molecules and coordination compounds. However, the ionization efficiency of some low polar molecules is low. And the situation is more serious in analysis of complex plant samples because of suppression effects of ionizable impurities. Signal fluctuation of mass spectrometry make accuracy and reproducibility poor in non-targeted metabolite determination. In vitro stable isotope labeling circumvented these problems, and has been used in relative quantitative determination of proteins and small metabolite. However, mixing of light and heavy labeled samples in stable isotope labeling protocol double the complexity of the samples. In this study, we try to solve the difficulties in plant metabolite analysis by using new synthesized isotope labeling reagent and new scanning method of mass spectrometry, including the following four subjects:Synthesis and Evaluation of New Derivatization Reagent With Quaternary Ammonium Moiety ?-bromoactonyltrimethylammonium bromide (BTA), ?-bromoacetonylpyridinium bromide (BPA) and co-bromoacetonylquinolinium bromide (BQA) were synthesized from reaction of trimethylamine, pyridine and quinoline with1,3-dibromoactone respectively. The three reagents shared the same reaction center, bromoacetonyl moiety. Experimental study and theoretical calculation showed that the reactivity increase gradually from BTA, BPA to BQA. The selectivities of the three reagents were further study. The results showed that BQA is a high selective reagent towards thiols, and the selectivity of BPA is higher than BTA towards carboxylic acid. The derivatization conditions of BPA with carboxylic acids and BQA with thiols were optimized. The labeling efficiencies and the improvement of ion signals were evaluated under the optimized conditions Use of Isotope Mass Probes for Metabolic Analysis of the Jasmonate Biosynthetic Pathway In order to quantitatively study the jasmonate biosynthetic pathway, we chemically synthesized a pair of isotope mass probes and established a labeling protocol. The pair of mass probes used in our work were ?-bromoacetonylpyridinium bromide (BPB) and d5-?-bromoacetonylpyridinium bromide (d5-BPB), which contain carboxyl acid reactive groups, isotopically labeled groups and permanent positive charges. High performance liquid chromatography (HPLC) and electrospray ionization Quadrupole-Time of Flight Mass Spectrometry (ESI-QTOF-MS) was used for the detection of labeled standard mixtures and plant samples. A data analysis method was established for analyzing metabolic pathways using our labeling strategy. We then applied our method and examined the jasmonate biosynthetic pathway of rice under salt stress and the premature senescence mutant. Here we found that under both conditions, significant changes were found in the biosynthetic pathway of jasmonate pathway.In Vitro Isotope Labeling-Double Neutral Loss Scan for Screening of Metabolite With Carboxyl Group In order to screen the metabolite with carboxyl group in wounded tomato seeds, we chemically synthesized a pair of isotope mass probes, including N,N-dimethylaminobutylamine and d4-N,N-dimethylaminobutylamine. This pair of mass probes used contain carboxyl acid reactive groups (amino moiety), isotopically labeled groups (methyl), ionizable moiety (tertiary amine). The isotope labeled dimethylamino moiety is easily loss as a neutral fragment in CID. In vitro isotope label ing-double neutral loss scan was performed in high performance liquid chromatography coupled with tandem mass spectrometry. The structures of pre-selected biomarkers were further confirmed by high resolution mass spectrometry and product ion scan. We discuss the biological functions of the potential biomarkers with the help of metabonomics databases and related literatures.Screening and Identification of Ribosides using In Vitro Isotope Labeling-Double Neutral Loss Scan Acetone can reacted selectively with neigbouring cis-dihydroxy and meta cis-dihydroxy group to generate more hydrophobic ketal. Glycosidic bonds are easily broken in CID, and the glycosyl moiety was loss as a neutral fragment. Aiming at no-targeted relative quantitative determination of ribosides, in vitro isotope labeling followed by HPLC-tandem MS double neutral loss scan was performed by using of acetone and d6-acetone as the labeling reagents. We screened the riboside changes in wounded tomato seeds. Compared with the control group, several riboside with significant different signal intensity were pre-selected. The structures of pre-selected biomarkers were further confirmed by high resolution mass spectrometry and product ion scan. We further discuss the biological functions of the potential biomarkers with the help of metabonomics databases and related literatures.