Global metabolic analyses of acinetobacter baumannii

Acinetobacter baumannii is rapidly emerging as a multidrug-resistant pathogen responsible for nosocomial infections including pneumonia, bacteremia, wound infections, urinary tract infections, and meningitis. Metabolomics provides a powerful tool to gain a system-wide snapshot of cellular biochemical networks under defined conditions and has been increasingly applied to bacterial physiology and drug discovery. Here we describe an optimized sample preparation method for untargeted metabolomics studies in A. baumannii. Our method provides a significant recovery of intracellular metabolites to demonstrate substantial differences in global metabolic profiles among A. baumannii strains

A batch correction method for liquid chromatography–mass spectrometry data that does not depend on quality control samples

The need for reproducible and comparable results is of increasing importance in non-targeted metabolomic studies, especially when differences between experimental groups are small. Liquid chromatography– mass spectrometry spectra are often acquired batch-wise so that necessary calibrations and cleaning of the instrument can take place. However this may introduce further sources of variation, such as differences in the conditions under which the acquisition of individual batches is performed. Quality control (QC) samples are frequently employed as a means of both judging and correcting this variation. Here we show that the use of QC samples can lead to problems. The non-linearity of the response can result in substantial differences between the recorded intensities of the QCs and experimental samples, making the required adjustment difficult to predict. Furthermore, changes in the response profile between one QC interspersion and the next cannot be accounted for and QC based correction can actually exacerbate the problems by introducing artificial differences. ‘‘Background correction’’ methods utilise all experimental samples to estimate the variation over time rather than relying on the QC samples alone. We compare non-QC correction methods with standard QC correction and demonstrate their success in reducing differences between replicate samples and their potential to highlight differences between experimental groups previously hidden by instrumental variation

Mass-spectrometry-based metabolomics: limitations and recommendations for future progress with particular focus on nutrition research

Mass spectrometry (MS) techniques, because of their sensitivity and selectivity, have become methods of choice to characterize the human metabolome and MS-based metabolomics is increasingly used to characterize the complex metabolic effects of nutrients or foods. However progress is still hampered by many unsolved problems and most notably the lack of well established and standardized methods or procedures, and the difficulties still met in the identification of the metabolites influenced by a given nutritional intervention. The purpose of this paper is to review the main obstacles limiting progress and to make recommendations to overcome them. Propositions are made to improve the mode of collection and preparation of biological samples, the coverage and quality of mass spectrometry analyses, the extraction and exploitation of the raw data, the identification of the metabolites and the biological interpretation of the results

Quantification of endogenous aminoacids and aminoacid derivatives in urine by hydrophilic interaction liquid chromatography tandem mass spectrometry

Aminoacids and their derivatives are key biologically important metabolites and reliable, rapid and accurate, quantification for these analytes in urine remains an important analytical challenge. Here a fast and reliable HILIC-tandem MS method is presented for application in clinical or nutritional studies. The developed method was validated according to existing guidelines adapted for endogenous analytes. The validation strategy provided evidence of linearity, LOD and LOQ, accuracy, precision, matrix effect and recovery. The surrogate matrix approach was applied for calibration proving satisfactory accuracy and precision based on standard criteria over the working concentration ranges. Intra and inter day accuracy was found to range between 0.8 and 20% for the LQC (low QC) and between 0.05 and 15 % for MQC (medium QC) and HQC (high QC). Inter and intraday precision were found to be between 3 and 20 % for the LQC and between 1 and 15% for the MQC and HQC. The stability of the analytes, in both surrogate and pooled urine QC samples, was found to be within 15% over a short period at 4 °C or after a up to 3 freeze-thaw cycles. The uncertainty of the method was also assessed to provide increased confidence for the acquired measurements. The method was successfully applied to a subset of human urine samples involved in a study of amino acids dietary uptake. This method may provide a valuable tool for many applications or studies where amino acid metabolic signatures in the excreted urine are under investigation

Hyphenated MS–Based Targeted approaches in metabolomics

While global metabolic profiling (untargeted metabolomics) has been the center of much interest and research activity in the past few decades, more recently targeted metabolomics approaches have begun to gain ground. These analyses are, to an extent, more hypothesis-driven, as they focus on a set of pre-defined metabolites and aim towards their determination, often to the point of absolute quantification. The continuous development of the technological platforms used in these studies facilitates the analysis of large numbers of well-characterized metabolites present in complex matrices. The present review describes recent developments in the hyphenated chromatographic methods most often applied in targeted metabolomic/lipidomic studies (LC-MS/MS, CE-MS/MS, and GC-MS/MS), highlighting applications in the life and food/plant sciences. The review also underlines practical challenges–limitations that appear in such approaches

Cutting-edge analytical technologies for the comprehensive metabolic profiling of Alkanna tinctoria roots cultured in greenhouse conditions

The use of plants containing naphthoquinone derivatives Alkannins & Shikonins (A/S) by humans dates back to ancient times. In recent decades, the use of A/S has seen a resurgence and A/S have risen to a pivotal role as pharmaceutical and cosmeceutical agents, since they possess strong wound healing, antimicrobial, anti-inflammatory, tissue regenerative and antitumor properties. It is thus crucial to enhance the biosynthesis of bioactive A/S in Alkanna tinctoria plants, that naturally produce high amounts of these metabolites [1]. In the frame of “MICROMETABOLITE” EU H2020 project, we have optimized a workflow for the metabolic profiling of A. tinctoria roots, cultured in the greenhouse from plants obtained by in vivo shoot cuttings. A fast and reliable extraction procedure was achieved for comprehensive profiling and identification of A/S and other metabolites biosynthesized in the roots. The aim of this work was to determine the growth stage with peak A/S production, while simultaneously obtaining additional information on the root metabolome. A combination of UHPLC-HRMS and NMR was used for metabolite identification, HPLC was utilized for reliable quantitation of A/S and the extracts were subjected to chiral HPLC analysis [2] for determination of the enantiomeric A/S ratio. Different A/S derivatives and other metabolites were identified in plant roots using UHPLC-HRMS and NMR. Six A/S derivatives and total A/S were quantified using HPLC-DAD. From six vegetation stages of A. tinctoria grown under greenhouse conditions, fruiting period was found to peak A/S production (1% wt/wt of root), while the enantiomeric alkannin/shikonin ratio remained constant (93.7%)