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Metabolomics is the field of omics research that offers valuable insights into the complex composition of biological samples. It has found wide application in clinical diagnostics, disease investigation, therapy prediction, monitoring of treatment efficiency, drug discovery, or in-depth analysis of sample composition. A suitable study design constitutes the fundamental requirements to ensure robust and reliable results from the study data. The study design process should include a careful selection of conditions for each experimental step, from sample collection to data analysis. The pre-analytical variability that can introduce bias to the subsequent analytical process, decrease the outcome reliability, and confuse the final results of the metabolomics research, should also be considered. Herein, we provide key information regarding the pre-analytical variables affecting the metabolomics studies of biological fluids that are the most desirable type of biological samples. Our work offers a practical review that can serve and guide metabolomics pre-analytical design. It indicates pre-analytical factors, which can introduce artificial data variation and should be identified and understood during experimental design (through literature overview or analytical experiments).

Human milk (HM) lipidome stability during storage is crucial in lipidomic studies to avoid misinterpretations. Facing the lack of comprehensive work on the HM lipidome stability, we performed a study on a potential alteration in the lipid profiles of HM samples stored under different conditions. An untargeted LC-Q-TOF-MS-based approach was applied to study the influence of storage conditions as well as the interaction of the storage temperature and time on HM lipid profiles. The samples were stored for 4-84 days at temperatures in the range from 4 to -80 °C and also were exposed to up to three freeze-thaw cycles. The results showed that the storage at 4 °C for just 4 days as well as being subjected to three freeze-thaw cycles can lead to a change in the content of lipids. The observed differences in levels of some lipid species in samples stored at -20 °C in comparison to the concentration level of those lipids in samples stored at -80 °C were not statistically significant, and inter-individual variance regardless of sample storage condition was maintained. The storage of HM samples at -20 °C for up to 3 weeks and -80 °C for up to 12 weeks ensures sample lipidome stability.

Human breast milk (HBM) is a biofluid consisting of various biomolecules such as proteins, lipids, carbohydrates, minerals and bioactive substances. Due to its unique and complex composition, HBM provides not only nutritional components required for the growth of the infant, but also additional protection against infections. Global insight into the composition of HBM is crucial to understanding the health benefits infants receive from breastfeeding and could be used to improve the composition of milk formula for babies that cannot be breastfed. To improve global profiling of the HBM lipidome, a new analytical approach based on solid-phase microextraction (SPME) and liquid chromatography-mass spectrometry (LC-MS) was developed. The new extraction method allows for the rapid and simple extraction of a broad range of lipids directly from HBM samples. Moreover, the optimized two-step lipid extraction protocol ensures high lipidome coverage without using toxic solvents such as chloroform. The use of liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) and an automated search of a lipid database allows comprehensive identification of the lipids contained in HBM. The demonstrated analytical approach based on SPME sample preparation and LC-Q-TOF-MS is rapid, free of toxic solvents, and suited for the qualitative analysis of the HBM lipid composition.

Cyanobacteria play an important role in several ecological environments, and they are widely accepted to be the ancestors of chloroplasts in modern plants and green algae. Cyanobacteria have become attractive models for metabolic engineering, with the goal of exploring them as microbial cell factories. However, the study of cyanobacterial lipids&rsquo, composition and variation, and the assessment of the lipids&rsquo, functional and structural roles have been largely overlooked. Here, we aimed at expanding the cyanobacterial lipidomic analytical pipeline by using an untargeted lipidomics approach. Thus, the lipid composition variation of the model cyanobacterium Synechocystis sp. PCC 6803 was investigated in response to both alternative cultivation setups and gene deletion. This approach allowed for detecting differences in total lipid content, alterations in fatty-acid unsaturation level, and adjustments of specific lipid species among the identified lipid classes. The employed method also revealed that the cultivation setup tested in this work induced a deeper alteration of the cyanobacterial cell lipidome than the deletion of a gene that results in a dramatic increase in the release of lipid-rich outer membrane vesicles. This study further highlights how growth conditions must be carefully selected when cyanobacteria are to be engineered and/or scaled-up for lipid or fatty acids production.

Early nutrition has an enormous influence on a child's physiological function, immune system maturation, and cognitive development. Human breast milk (HBM) is recognized as the gold standard for human infant nutrition. According to a WHO report, breastfeeding is considered as an unequaled way of providing ideal food to the infant, which is required for his healthy growth and development. HBM contains various macronutrients (carbohydrates, proteins, lipids, and vitamins) as well as numerous bioactive compounds and interactive elements (growth factors, hormones, cytokines, chemokines, and antimicrobial compounds. The aim of this review is to summarize and discuss the current knowledge about metabolites, which are the least understood components of HBM, and their potential role in infant development. We focus on small metabolites (

Staphylococcus aureus is a common pathogen known to cause relatively minor infections as well as severe disorders in humans. Although there is fair amount of published data concerning various aspects of its biology, epidemiology, genetics, etc., there is still a scarce amount of data presenting reliable and thorough investigations regarding high-throughput analysis of total S. aureus lipid content. Therefore, the aim of this study was to develop an analytical method that in combination with advanced chemometric tools enables comprehensive lipidomic analysis of S. aureus cells. The newly developed method uses high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) to directly examine extracted lipids and further identify them within a newly developed novel S. aureus lipids database. High coverage of the S. aureus lipidome was obtained by simultaneous bacterial cell lysis and liquid-liquid extraction. The combination of three techniques enabled the analysis of the major membrane lipid classes of S. aureus: separation of the lipid extract in reversed-phase mode, Q-TOF-MS detection in positive ion mode, and lipid database mining. The developed lipidomic approach is also a powerful tool that allows one to assess the lipid content of S. aureus cells in a comparative manner between strains characterized by different phenotypic features as exemplified here by their various sensitivities toward antibiotics.

Staphylococcus aureus resistance to antibiotics is a significant clinical problem worldwide. In this study, an untargeted lipidomics approach was used to compare the lipid fingerprints of S. aureus clinical isolates that are resistant and sensitive to antibiotics. High-performance liquid chromatography coupled with time-of-flight mass spectrometry was employed to rapidly and comprehensively analyze bacterial lipids. Chemometric and statistical analyses of the obtained lipid fingerprints revealed variations in several lipid groups between S. aureus strains resistant and sensitive to tested antibiotics including methicillin, gentamicin, ciprofloxacin, erythromycin, and fusidic acid. The levels of identified monoglycosyldiacylglycerol, phosphatidylglycerol, and diglycosyldiacylglycerol lipid groups were found to be upregulated in antibiotic-resistant S. aureus strains, whereas the levels of diacylglycerol lipid groups were downregulated. Differences in the lipid patterns between sensitive and resistant S. aureus strains suggest that antibiotic susceptibility may be associated with the lipid composition of bacterial cells. The lipids that were found to significantly differ between antibiotic-resistant and antibiotic-sensitive clinical isolates are involved in the biosynthesis of major S. aureus membrane lipids and lipoteichoic acid. This study indicates that S. aureus lipid biosynthesis pathways should be explored further to better understand the mechanism of antibiotic resistance in S. aureus strains.

A set of 15 new sulphonamide derivatives, presenting antitumor activity have been subjected to a metabolic stability study. The results showed that besides products of biotransformation, some additional peaks occurred in chromatograms. Tandem mass spectrometry revealed the same mass and fragmentation pathway, suggesting that geometric isomerization occurred. Thus, to support this hypothesis, quantitative structure-retention relationships were applied. Human liver microsomes were used as an in vitro model of metabolism. The biotransformation reactions were tracked by liquid chromatography assay and additionally, fragmentation mass spectra were recorded. In silico molecular modeling at a semi-empirical level was conducted as a starting point for molecular descriptor calculations. A quantitative structure-retention relationship model was built applying multiple linear regression based on selected three-dimensional descriptors. The studied compounds revealed high metabolic stability, with a tendency to form hydroxylated biotransformation products. However, significant chemical instability in conditions simulating human body fluids was noticed. According to literature and MS data geometrical isomerization was suggested. The developed in sillico model was able to describe the relationship between the geometry of isomer pairs and their chromatographic retention properties, thus it supported the hypothesis that the observed pairs of peaks are most likely geometric isomers. However, extensive structural investigations are needed to fully identify isomers’ geometry. An effort to describe MS fragmentation pathways of novel chemical structures is often not enough to propose structures of potent metabolites and products of other chemical reactions that can be observed in compound solutions at early drug discovery studies. The results indicate that the relatively non-expensive and not time- and labor-consuming in sillico approach could be a good supportive tool assisting the identification of cis-trans isomers based on retention data. This methodology can be helpful during the structural identification of biotransformation and degradation products of new chemical entities - potential new drugs.

Light- and photosensitiser-based antimicrobial photodynamic therapy is a very promising approach to the control of microbial infections. How the phenotypic features of a microorganism affect its response to photosensitiser-based photokilling represents an area of substantial research interest. To understand the mechanisms governing the phenomenon of a strain-dependent response to photodynamic inactivation (PDI), we analysed the possible role of the membrane-located haem transporter HrtA in Staphylococcus aureus. We used a S. aureus strains with an inactivated component of the haem-regulated transporter, HrtA, along with its wild-type counterpart to determine differences in PDI outcome and photosensitiser uptake between the studied isogenic strains. We observed that a lack of HrtA protein potentiates the phototoxic effect towards S. aureus but only when extracellular protoporphyrin IX is used. The observed effect may depend on the function of the HrtA transporter but is likely to result from changed membrane properties following the absence of the protein in the membrane. This indicates that disturbing the membrane properties is an attractive method for improving the efficacy of the photodynamic inactivation of microorganisms. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7145-5) contains supplementary material, which is available to authorized users.