The dual trend in histatins research

We review histatins (Hsts) in order to encourage development of analytical platforms to clarify obscure points in knowledge of this family of antimicrobial and antifungal peptides. To explain the present interest, we outline the number and the nature of Hsts and their known functions (i.e. antimicrobial action, wound closure, biomarkers of stress, satiety, body mass and incipient Alzheimer’s disease, and diagnosis and treatment of addiction, including to cocaine).The two aspects of research on Hsts (i.e. their natural effects on living organisms and their potential use for medical applications, including as biomarkers) make it necessary to develop new analytical methods. The variety of matrices in which Hsts exist (e.g., saliva and tooth-surface-protein pellicle) make it essential to develop new sample-preparation steps and to improve identification and quantitation steps as analytical instrumentation evolves.In this context, metabolomics studies could be of great interest, as contributions of analytical chemists could be one of the keys to achieving the role that they deserve within “-omics” research.     

CE‐MS for metabolomics: Developments and applications in the period 2016–2018

In the field of metabolomics, CE‐MS is now recognized as a strong analytical technique for the analysis of (highly) polar and charged metabolites in a wide range of biological samples. Over the past few years, significant attention has been paid to the design and improvement of CE‐MS approaches for (large‐scale) metabolic profiling studies and for establishing protocols in order to further expand the role of CE‐MS in metabolomics. In this paper, which is a follow‐up of a previous review paper covering the years 2014–2016 (Electrophoresis 2017, 38, 190–202), main advances in CE‐MS approaches for metabolomics studies are outlined covering the literature from July 2016 to June 2018. Aspects like developments in interfacing designs and data analysis tools for improving the performance of CE‐MS for metabolomics are discussed. Representative examples highlight the utility of CE‐MS in the fields of biomedical, clinical, microbial, and plant metabolomics. A complete overview of recent CE‐MS‐based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings and MS detection mode. Finally, some general conclusions and perspectives are given.     

Development of a solid phase extraction protocol coupled with liquid chromatography mass spectrometry to analyze central carbon metabolites in lake sediment microcosms

An ion exchange solid phase extraction (SPE) strategy is developed for application with liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) for characterization of central carbon metabolites involved in methane assimilation and adjacent pathways in natural mixtures. For this purpose, short-time microcosm samples were obtained from lake sediment known to consume methane. Three SPE procedures were developed for the recovery of 51 targeted metabolites from five compound classes (amino acids, carboxylic acids, sugar phosphates, nucleotides and acyl-CoAs). The three SPE procedures employed were mixed mode (i) strong cation exchange, (ii) strong anion exchange and (iii) weak anion exchange. By spiking stable isotopic labeled standards, validation of the SPE procedures for the sediment extracts demonstrated that a 3 cm(3), 60 mg SPE sorbent bed provided effective loading capacity for targeted metabolites with an analytical variation of 16% RSD. We readily analyzed 32 of the targeted 51 metabolites using LC-MS/MS after sediment sample extraction, cleanup and pre-concentration. The remaining 19 targeted metabolites were either at, or below, the limit of detection. The current approach provides a good workflow for absolute quantification of intermediates in C(1)-carbon metabolism in natural microbial communities.     

Comparison of different liquid chromatography stationary phases in LC-HRMS metabolomics for the detection of recombinant growth hormone doping control

Growth hormone (GH) is a polypeptide suspected of being used in horse racing to speed up physical performances. Despite scientific advances in the recent years, the control of its administration remains difficult. In order to improve it, a metabolomics study through LC-high resolution mass spectrometry measurements was recently initiated to assess the metabolic perturbations caused by recombinant equine growth hormone administration. Few tens of ions not identified structurally were highlighted as compounds responsible for the modification of metabolic profiling observed in treated animals. This previous work was based on the use of Uptisphere Strategy NEC as the chromatographic column. In parallel, more and more metabolomics studies showed the interest of the use of new chromatographic supports such as hydrophilic interaction chromatography for the analysis of polar compounds. It is in this context that an investigation was conducted on Uptisphere HDO and Luna hydrophilic interaction chromatography stationary phases to generate and process urinary metabolomics fingerprints, which could allow to establish a comparison with Uptisphere Strategy NEC. The chromatographic column the most adapted for the detection of new biomarkers of GH administration has been used to set up a relevant statistical model based on the analysis of more than hundred biological samples.     

Ultra-performance liquid chromatography coupled to mass spectrometry as a sensitive and powerful technology for metabolomic studies

Metabolomics is the comprehensive assessment of endogenous metabolites of a biological system. These large-scale analyses of metabolites are intimately bound to advancements in ultra-performance liquid chromatography-electrospray (UPLC) technologies and have emerged in parallel with the development of novel mass analyzers and hyphenated techniques. Recently, the combination of UPLC with MS covers a number of polar metabolites, thus enlarging the number of detected analytes in the widely used separation sciences. This technology has rapidly been accepted by the analytical community and is being gradually applied to various fields such as metabolomics and traditional Chinese medicine (TCM). Given the power of the technology, metabolomics has become increasingly popular in drug development, molecular medicine, traditional medicine and other biotechnology fields, since it profiles directly the phenotype and changes thereof in contrast to other "-omics" technologies. Hyphenated UPLC/MS technique is becoming a useful tool in the study of body fluids, represents a promising hyphenated microseparation platform in metabolomics and has a strong potential to contribute to disease diagnosis. This review describes the applications of UPLC/MS in metabolomic research, and comparison role of HPLC/MS, NMR and GC/MS, highlights its advantages and limitations with certain characteristic examples in the life and TCM sciences.     

Liquid–liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids

A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid–liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The ¹²C-/¹³C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC–FTICR–MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC–MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10 μL of urine. We believe that this method opens the possibility of generating a very comprehensive profile of the organic acid sub-metabolome that will be useful for comparative metabolomics applications for biological studies and disease biomarker discovery.     

Simultaneous quantification of energetically important metabolites in various cell types by CZE

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field‐enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on‐line pre‐concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.     

Mass spectrometry-based metabolomics applied to the chemical safety of food

Mass spectrometry (MS)-based metabolomics is emerging as an important field of research in many scientific areas, including chemical safety of food. A particular strength of this approach is its potential to reveal some physiological effects induced by complex mixtures of chemicals present at trace concentrations. The limitations of other analytical approaches currently employed to detect low-dose and mixture effects of chemicals make detection very problematic. Besides this basic technical challenge, numerous analytical choices have to be made at each step of a metabolomics study, and each step can have a direct impact on the final results obtained and their interpretation (i.e. sample preparation, sample introduction, ionization, signal acquisition, data processing, and data analysis). As the application of metabolomics to chemical analysis of food is still in its infancy, no consensus has yet been reached on defining many of these important parameters. In this context, the aim of the present study is to review all these aspects of MS-based approaches to metabolomics, and to give a comprehensive, critical overview of the current state of the art, possible pitfalls, and future challenges and trends linked to this emerging field.     

Metabolite profiling on apple volatile content based on solid phase microextraction and gas-chromatography time of flight mass spectrometry

A headspace SPME GC-TOF-MS method was developed for the acquisition of metabolite profiles of apple volatiles. As a first step, an experimental design was applied to find out the most appropriate conditions for the extraction of apple volatile compounds by SPME. The selected SPME method was applied in profiling of four different apple varieties by GC-EI-TOF-MS. Full scan GC-MS data were processed by MarkerLynx software for peak picking, normalisation, alignment and feature extraction. Advanced chemometric/statistical techniques (PCA and PLS-DA) were used to explore data and extract useful information. Characteristic markers of each variety were successively identified using the NIST library thus providing useful information for variety classification. The developed HS-SPME sampling method is fully automated and proved useful in obtaining the fingerprint of the volatile content of the fruit. The described analytical protocol can aid in further studies of the apple metabolome.