Characterization of trichome-specific BAHD acyltransferases involved in acylsugar biosynthesis in Nicotiana tabacum.

Glandular trichomes of tobacco (Nicotiana tabacum) produce blends of acylsucroses that contribute to defence against pathogens and herbivorous insects, but the mechanism of assembly of these acylsugars has not yet been determined. In this study, we isolated and characterized two trichome-specific acylsugar acyltransferases that are localized in the endoplasmic reticulum, NtASAT1 and NtASAT2. They sequentially catalyse two additive steps of acyl donors to sucrose to produce di-acylsucrose. Knocking out of NtASAT1 or NtASAT2 resulted in deficiency of acylsucrose; however, there was no effect on acylsugar accumulation in plants overexpressing NtASAT1 or NtASAT2. Genomic analysis and profiling revealed that NtASATs originated from the T subgenome, which is derived from the acylsugar-producing diploid ancestor N. tomentosiformis. Our identification of NtASAT1 and NtASAT2 as enzymes involved in acylsugar assembly in tobacco potentially provides a new approach and target genes for improving crop resistance against pathogens and insects.

Identification of chloramination disinfection by-products from phenylalanine in tap drinking water.

Phenylalanine (Phe) is widely present in natural water and serves as a precursor of disinfection by-products (DBPs). We reported the identification of chloramination DBPs from Phe in drinking water using ultra-high performance liquid chromatography (UHPLC) coupled with complementary high-resolution quadrupole time-of-flight (QTOF) and triple quadrupole (tQ) tandem mass spectrometry (MS/MS). In the chloraminated Phe water solution, sixteen new DBPs in a total of seventeen were identified based on their accurate mass, MS/MS spectra and 35Cl/37Cl isotopic patterns. Three of these DBPs were verified as benzamide, phenylacetamide, and p-hydroxyphenylacetamide with their standards, while the others were chlorinated derivatives of Phe, hydrazone, amidine, amide and peroxide, in which the unique structures of these DBPs were rarely reported. Their stability and formation process were investigated as well. Furthermore, a method consisting of solid phase extraction (SPE) and UHPLC-MS/MS using dynamic multiple reaction monitoring (dMRM) was developed to investigate these DBPs in authentic waters. Phe, benzamide, phenylacetamide, and N-Cl-2-phenylacetimidamide were detected in chlorinated tap water. Compared with the other identified DBPs, these three DBPs were exceptionally stable and could be formed in wide formation conditions. Our work not only provided ideas for the identification of new chloramination DBPs, but also demonstrated that some DBPs usually generated in the chloramination disinfection process could also be found in the chlorinated drinking water.

Identification of new trace related impurities in adenosine products using ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: Adenosine can induce various physiopathological effects and has been adopted as a drug to treat certain forms of supraventricular tachycardia. Adenosine is predominantly produced via fermentation that may generate many other bioactive compounds with similar structures at trace levels. Thus, it is necessary to identify these trace structurally related impurities. A new method using ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) is proposed to separate complex components at trace amounts; to obtain accurate mass measurements providing the elemental composition of unknown compounds; and MS/MS spectra for elucidation of structures. METHODS: A UHPLC/QTOF-MS method was developed to separate and detect trace related impurities with structures similar to adenosine in formation samples. MS and MS/MS spectra of the impurities detected in real samples were acquired and used to propose their structures. Available reference standards were used to confirm the identification of some of the impurities detected. RESULTS: Nine trace impurities of adenosine were separated and characterized. Of these nine, five were confirmed as 5'-adenylic acid, hypoxanthine, inosine, adenine, and 2'-deoxyadenosine by comparison with their available reference standards. The remaining four were proposed to be ADP-ribose, two S-epimers of 5'-deoxy-5'-methylsulfinyl adenosine, and 3'-α-glucosyl adenosine based on their MS and MS/MS spectra, and available literature. Additionally, the MS/MS fingerprints of the monosaccharide glycosyl groups were discovered and discussed. CONCLUSIONS: A UHPLC/QTOF-MS method was established and used for the separation and characterization of nine trace related impurities of adenosine. Their structures were identified based on the MS and MS/MS spectra and retention times. In addition, the specific MS/MS fingerprints of the monosaccharide glycosyl moieties of the adenosine impurity analogs were summarized.