Gas Chromatography-Mass Spectrometry (GC-MS) has long been used for metabolite profiling of a wide range of biological samples. Many derivatisation protocols are already available and among these, trimethylsilyl (TMS) derivatisation is one of the most widely used in metabolomics. However, most TMS methods rely on off-line derivatisation prior to GC-MS analysis. In the case of manual off-line TMS derivatisation, the derivative created is unstable, so reduction in recoveries occurs over time. Thus, derivatisation is carried out in small batches. Here, we present a fully automated TMS derivatisation protocol using robotic autosamplers and we also evaluate a commercial software, Maestro available from Gerstel GmbH. Because of automation, there was no waiting time of derivatised samples on the autosamplers, thus reducing degradation of unstable metabolites. Moreover, this method allowed us to overlap samples and improved throughputs. We compared data obtained from both manual and automated TMS methods performed on three different matrices, including standard mix, wine, and plasma samples. The automated TMS method showed better reproducibility and higher peak intensity for most of the identified metabolites than the manual derivatisation method. We also validated the automated method using 114 quality control plasma samples. Additionally, we showed that this online method was highly reproducible for most of the metabolites detected and identified (RSD < 20) and specifically achieved excellent results for sugars, sugar alcohols, and some organic acids. To the very best of our knowledge, this is the first time that the automated TMS method has been applied to analyse a large number of complex plasma samples. Furthermore, we found that this method was highly applicable for routine metabolite profiling (both targeted and untargeted) in any metabolomics laboratory.

Th e colourful appearance of avian eggshells is a prominent aspect of maternal reproductive eff ort in birds. Some diff erences in eggshell coloration have been reported to co-vary with various measures of maternal condition and these patterns support the hypothesis that, in some bird species, several aspects of eggshell colour (i.e. primary chroma and brightness) function as a signal of maternal and off spring quality to induce greater paternal investment. We directly quantifi ed eggshell pigment concentrations of blackbird Turdus merula and song thrush T. philomelos eggshells and tested how the two key pigments (protoporphyrin IX and biliverdin) co-varied with other eggshell traits and egg constituents as measures of maternal reproductive investment, including total yolk carotenoid concentration, total lipid concentration, yolk mass, and shell thickness. Contrary to predictions, we detected few statistical patterns overall. We found that protoporphyrin IX concentration was negatively associated with blue-green chroma in blackbirds but not in song thrush. Th e concentration of protoporphyrin IX was signifi cantly greater in blackbirds and also showed diff erent patterns of association with total yolk lipids and yolk carotenoid concentrations between these two species (signifi cant species interaction terms). Our results reveal that it is not appropriate to simply assume in these two avian species that refl ectancebased eggshell colour measures are a suitable proxy for eggshell pigment concentrations or can be used as consistent predictors of maternal reproductive investment. Th ese results highlight the need to assess and validate the strength and direction of the statistical relationships between eggshell colour measures, pigment concentrations, and maternal resource deposition in the egg for other species of birds.

The antimicrobial role of itaconic acid (ITA) has been recently discovered in mammalian cells. In our previous studies, we discovered that marine molluscs biosynthesise substantial quantities of ITA when exposed to marine pathogens, but its antimicrobial function to Vibrio bacteria is currently unknown. Thus, in this study, we used an untargeted gas chromatography–mass spectrometry (GC-MS) platform to identify metabolic changes of Vibrio sp. DO1 ( V . corallyliticus / neptunius -like isolate) caused by ITA exposure. Vibrio sp. DO1 was cultured in Luria-Bertani broth supplemented with 3 mM sodium acetate and with different concentrations of ITA (0, 3 and 6 mM) for 24 h. The results showed that ITA completely inhibited Vibrio sp. growth at 6 mM and partially inhibited the bacterial growth at 3 mM. A principal component analysis (PCA) revealed a clear separation between metabolite profiles of Vibrio sp. DO1 in the 3 mM ITA treatment and the control, which were different in 25 metabolites. Among the altered metabolites, the accumulation of glyoxylic acid and other metabolites in glyoxylate cycle (cis-aconitic acid, isocitric acid and fumaric acid) together with the increase of isocitrate lyase (ICL) activity in the 3 mM ITA treatment compared to the control suggest that ITA inhibited Vibrio sp. growth via disruption of central carbon metabolism.

No abstract

No abstract