Hyphenation of microflow chromatography with electrospray ionization mass spectrometry for bioanalytical applications focusing on low molecular weight compounds: A tutorial review.

Benefits of miniaturized chromatography with various detection modes, such as increased sensitivity, chromatographic efficiency, and speed, were recognized nearly 50 years ago. Over the past two decades, this approach has experienced rapid growth, driven by the emergence of mass spectrometry applications serving -omics sciences and the need for analyzing minute volumes of precious samples with ever higher sensitivity. While nanoscale liquid chromatography (flow rates <1 µL/min) has gained widespread recognition in proteomics, the adoption of microscale setups (flow rates ranging from 1 to 100 µL/min) for low molecular weight compound applications, including metabolomics, has been surprisingly slow, despite the inherent advantages of the approach. Highly heterogeneous matrices and chemical structures accompanied by a relative lack of options for both selective sample preparation and user-friendly equipment are usually reported as major hindrances. To facilitate the wider implementation of microscale analyses, we present here a comprehensive tutorial encompassing important theoretical and practical considerations. We provide fundamental principles in micro-chromatography and guide the reader through the main elements of a microflow workflow, from LC pumps to ionization devices. Finally, based on both our literature overview and experience, illustrated by some in-house data, we highlight the critical importance of the ionization source design and its careful optimization to achieve significant sensitivity improvement.

Low-polarity untargeted metabolomic profiling as a tool to gain insight into seminal fluid.

INTRODUCTION: A decrease in sperm cell count has been observed along the last several decades, especially in the most developed regions of the world. The use of metabolomics to study the composition of the seminal fluid is a promising approach to gain access to the molecular mechanisms underlying this fact. OBJECTIVES: In the present work, we aimed at relating metabolomic profiles of young healthy men to their semen quality parameters obtained from conventional microscopic analysis. METHODS: An untargeted metabolomics approach focusing on low- to mid-polarity compounds was used to analyze a subset of seminal fluid samples from a cohort of over 2700 young healthy men. RESULTS: Our results show that a broad metabolic profiling comprising several families of compounds (including acyl-carnitines, steroids, and other lipids) can contribute to effectively distinguish samples provided by individuals exhibiting low or high absolute sperm counts. CONCLUSION: A number of metabolites involved in sexual development and function, signaling, and energy metabolism were highlighted as being distinctive of samples coming from either group, proving untargeted metabolomics as a promising tool to better understand the pathophysiological processes responsible for male fertility impairment.

Bioactive Nitrosylated and Nitrated N-(2-hydroxyphenyl)acetamides and Derived Oligomers: An Alternative Pathway to 2-Amidophenol-Derived Phytotoxic Metabolites.

Incubation of Aminobacter aminovorans, Paenibacillus polymyxa, and Arthrobacter MPI764 with the microbial 2-benzoxazolinone (BOA)-degradation-product 2-acetamido-phenol, produced from 2-aminophenol, led to the recently identified N-(2-hydroxy-5-nitrophenyl) acetamide, to the hitherto unknown N-(2-hydroxy-5-nitrosophenyl)acetamide, and to N-(2-hydroxy-3-nitrophenyl)acetamide. As an alternative to the formation of phenoxazinone derived from aminophenol, dimers- and trimers-transformation products have been found. Identification of the compounds was carried out by LC/HRMS and MS/MS and, for the new structure N-(2-hydroxy-5-nitrosophenyl)acetamide, additionally by 1D- and 2D-NMR. Incubation of microorganisms, such as the soil bacteria Pseudomonas laurentiana, Arthrobacter MPI763, the yeast Papiliotrema baii and Pantoea ananatis, and the plants Brassica oleracea var. gongylodes L. (kohlrabi) and Arabidopsis thaliana Col-0, with N-(2-hydroxy-5-nitrophenyl) acetamide, led to its glucoside derivative as a prominent detoxification product; in the case of Pantoea ananatis, this was together with the corresponding glucoside succinic acid ester. In contrast, Actinomucor elegans consortium synthesized 2-acetamido-4-nitrophenyl sulfate. 1 mM bioactive N-(2-hydroxy-5-nitrophenyl) acetamide elicits alterations in the Arabidopsis thaliana expression profile of several genes. The most responsive upregulated gene was pathogen-inducible terpene synthase TPS04. The bioactivity of the compound is rapidly annihilated by glucosylation.

Anti-microtubule activity of the traditional Chinese medicine herb Northern Ban Lan (Isatis tinctoria) leads to glucobrassicin.

Traditional Chinese medicine (TCM) belongs to the most elaborate and extensive systems of plant-based healing. The herb Northern Ban Lan (Isatis tinctoria) is famous for its antiviral and anti-inflammatory activity. Although numerous components isolated from I. tinctoria have been characterized so far, their modes of action have remained unclear. Here, we show that extracts from I. tinctoria exert anti-microtubular activity. Using time-lapse microscopy in living tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cells expressing green fluorescent protein-tubulin, we use activity-guided fractionation to screen out the biologically active compounds of I. tinctoria. Among 54 fractions obtained from either leaves or roots of I. tinctoria by methanol (MeOH/H2 O 8:2), or ethyl acetate extraction, one specific methanolic root fraction was selected, because it efficiently and rapidly eliminated microtubules. By combination of further purification with ultra-high-performance liquid chromatography and high-resolution tandem mass spectrometry most of the bioactivity could be assigned to the glucosinolate compound glucobrassicin. Glucobrassicin can also affect microtubules and induce apoptosis in HeLa cells. In the light of these findings, the antiviral activity of Northern Ban Lan is discussed in the context of microtubules being hijacked by many viral pathogens for cell-to-cell spread.

Comprehensive plasma steroidomics reveals subtle alterations of systemic steroid profile in patients at different stages of prostate cancer disease.

The steroid submetabolome, or steroidome, is of particular interest in prostate cancer (PCa) as the dependence of PCa growth on androgens is well known and has been routinely exploited in treatment for decades. Nevertheless, the community is still far from a comprehensive understanding of steroid involvement in PCa both at the tissue and at systemic level. In this study we used liquid chromatography/high resolution mass spectrometry (LC/HRMS) backed by a dynamic retention time database DynaSTI to obtain a readout on circulating steroids in a cohort reflecting a progression of the PCa. Hence, 60 relevant compounds were annotated in the resulting LC/HRMS data, including 22 unknown steroid isomers therein. Principal component analysis revealed only subtle alterations of the systemic steroidome in the study groups. Next, a supervised approach allowed for a differentiation between the healthy state and any of the stages of the disease. Subsequent clustering of steroid metabolites revealed two groups responsible for this outcome: one consisted primarily of the androgens, whereas another contained corticosterone and its metabolites. The androgen data supported the currently established involvement of a hypothalamic-pituitary-gonadal axis in the development of PCa, whereas biological role of corticosterone remained elusive. On top of that, current results suggested a need for improvement in the dynamic range of the analytical methods to better understand the role of low abundant steroids, as the analysis revealed an involvement of estrogen metabolites. In particular, 2-hydroxyestradiol-3-methylether, one of the compounds present in the disease phenotype, was annotated and reported for the first time in men.

Investigation of several chromatographic approaches for untargeted profiling of central carbon metabolism.

Monitoring the central carbon metabolism (CCM) network using liquid chromatography/mass spectrometry (LC-MS) analysis is hampered by the diverse chemical nature of its analytes, which are extremely difficult to analyze using single chromatographic conditions. Furthermore, CCM-related compounds present non-specific adsorption on metal surfaces, causing detrimental chromatographic effects and sensitivity loss. In this study, polar reversed-phase, mixed-mode (MMC), and zwitterionic hydrophilic interaction chromatography (HILIC) featuring low-adsorption hardware were investigated towards untargeted analysis of biological samples with a focus on energy metabolism-related analytes. Best results were achieved with sulfoalkylbetaine HILIC with different supports, where polymeric option featured the highest coverage and inert hybrid silica facilitated best throughput and kinetic performance at a cost of less selectivity for small carboxylic acids. MMC demonstrated excellent performance for strongly anionic analytes such as multiresidue phosphates. The obtained experimental data also suggested that an additional hydrophilic modulation might be necessary to facilitate better resolution of carboxylic acids in zHILIC mode, as found during the application of the developed method to study the effect of two different mutations on the energy metabolism of S. aureus.

Separation and determination of cysteine enantiomers in plasma after derivatization with 4-fluoro-7-nitrobenzofurazan.

The importance of D-amino acids in mammals associated with enantio-dependent biological functions has been increasingly highlighted. In addition to naturally occurring, D-amino acid supplementation could have a positive biological impact, including cytoprotective implications. In this context, supplementation with D-cysteine has revealed beneficial effects. Quantification of cysteine enantiomers in rodent plasma has been achieved by using 4-fluoro-7-nitrobenzofurazan derivatization of the target analytes. Cystine, the main form of cysteine in the plasma, was initially reduced to cysteine using DL-dithiothreitol. Baseline enantioseparation was then achieved in less than 3 min using a (R,R)-Whelk-O 1 stationary phase and isocratic elution using CH3OH-H2O 90:10 (v/v) with 15 mM ammonium formate (apparent pH 6.0) at 0.5 mL/min. The derivatives were then detected using negative ESI-MS in SRM mode. An external calibration was employed for D-cysteine, while L-cysteine quantification, as an endogenous analyte, was addressed using a background subtraction strategy. The method was validated. Response functions were obtained from 0 to 300 µM and from 0 to 125 µM for D-cysteine and L-cysteine, respectively. The trueness ranged from 96% to 105% for both enantiomers with repeatability and intermediate precision lower than 8% and 15% for the D-form and the endogenous L-form, respectively. The method was successfully applied for determining D- and L-cysteine in mouse plasma after D-cysteine administration.

Teg58, a small regulatory RNA, is involved in regulating arginine biosynthesis and biofilm formation in Staphylococcus aureus.

Staphylococcus aureus adapts to different environments by sensing and responding to diverse environmental cues. The responses are coordinately regulated by regulatory proteins, and small regulatory RNAs at the transcriptional and translational levels. Here, we characterized teg58, a SarA repressed sRNA, using ChIP-Seq and RNA-Seq analysis of a sarA mutant. Phenotypic and genetic analyses indicated that inactivation of teg58 led to reduced biofilm formation in a process that is independent of SarA, agr, PIA, and PSMs. RNA-Seq analysis of teg58 mutant revealed up-regulation of arginine biosynthesis genes (i.e., argGH) as well as the ability of the mutant to grow in a chemical defined medium (CDM) lacking L-arginine. Exogenous L-arginine or endogenous induction of argGH led to decreased biofilm formation in parental strains. Further analysis in vitro and in vivo demonstrated that the specific interaction between teg58 and the argGH occurred at the post-transcriptional level to repress arginine synthesis. Biochemical and genetic analyses of various arginine catabolic pathway genes demonstrated that the catabolic pathway did not play a significant role in reduced biofilm formation in the teg58 mutant. Overall, results suggest that teg58 is a regulatory sRNA that plays an important role in modulating arginine biosynthesis and biofilm formation in S. aureus.