Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

Phenolic composition and bioactivity of Ribes magellanicum fruits from southern Patagonia.

Eight Ribes magellanicum collections from three different places in southern Patagonia were compared for content of different groups of phenolics, antioxidant capacity and inhibition of enzymes related to metabolic syndrome (α-amylase, α-glucosidase and pancreatic lipase). The sample with the highest antioxidant capacity was assessed for glutathione (GSH) synthesis stimulation in human gastric adenocarcinoma (AGS) cells. The chemical profile was determined by high performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) and the main phenolics were quantified. The samples from Navarino Island and Reserva Nacional Magallanes showed higher content of anthocyanins and caffeoylquinic acid, with better activity towards α-glucosidase and antioxidant capacity. A sample from Omora (Navarino Island), significantly increased intracellular GSH content in AGS cells. Some 70 compounds were identified in the fruit extracts by HPLC-MS/MS. The glucoside and rutinoside from delphinidin and cyanidin and 3-caffeoylquinic acid were the main compounds. Different chemical profiles were found according to the collection places.

Urinary Glycosaminoglycans Are Associated with Recurrent UTI and Urobiome Ecology in Postmenopausal Women.

Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units of uronic acid and amino sugars. The luminal surface of the bladder epithelium is coated with a GAG layer. These urothelial GAGs are thought to provide a protective barrier and serve as a potential interaction site with the urinary microbiome (urobiome). Previous studies have profiled urinary GAG composition in mixed cohorts, but the urinary GAG composition in postmenopausal women remains undefined. To investigate the relationship between GAGs and recurrent urinary tract infection (rUTI), we profiled urinary GAGs in a controlled cohort of postmenopausal women. We found that chondroitin sulfate (CS) is the major urinary GAG in postmenopausal women and that urinary CS was elevated in women with active rUTI. We also associated urinary GAGs with urobiome composition and identified bacterial species that significantly associated with urinary GAG concentration. Corynebacterium amycolatum, Porphyromonas somerae, and Staphylococcus pasteuri were positively associated with heparin sulfate or hyaluronic acid, and bacterial species associated with vaginal dysbiosis were negatively correlated with urinary CS. Altogether, this work defines changes in urinary GAG composition associated with rUTI and identifies new associations between urinary GAGs and the urobiome that may play a role in rUTI pathobiology.

Urinary Glycosaminoglycans are Associated with Recurrent UTI and Urobiome Ecology in Postmenopausal Women.

Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units of uronic acid and amino sugars. The luminal surface of the bladder epithelium is coated with a GAG layer. These urothelial GAGs are thought to provide a protective barrier and serve as a potential interaction site with the urinary microbiome (urobiome). Previous studies have profiled urinary GAG composition in mixed cohorts, but the urinary GAG composition in postmenopausal women remains undefined. To investigate the relationship between GAGs and recurrent UTI (rUTI), we profiled urinary GAGs in a controlled cohort of postmenopausal women. We found that chondroitin sulfate (CS) is the major urinary GAG in postmenopausal women and that urinary CS was elevated in women with active rUTI. We also associated urinary GAGs with urobiome composition and identified bacterial species that significantly associated with urinary GAG concentration. Corynebacterium amycolatum, Porphyromonas somerae , and Staphylococcus pasteuri were positively associated with heparin sulfate or hyaluronic acid and bacterial species associated with vaginal dysbiosis were negatively correlated to urinary CS. Altogether, this work defines changes in urinary GAG composition associated with rUTI and identifies new associations between urinary GAGs and the urobiome that may play a role in rUTI pathobiology.

Recurrent urinary tract infection and estrogen shape the taxonomic ecology and function of the postmenopausal urogenital microbiome.

Postmenopausal women are severely affected by recurrent urinary tract infection (rUTI). The urogenital microbiome is a key component of the urinary environment. However, changes in the urogenital microbiome underlying rUTI susceptibility are unknown. Here, we perform shotgun metagenomics and advanced culture on urine from a controlled cohort of postmenopausal women to identify urogenital microbiome compositional and function changes linked to rUTI susceptibility. We identify candidate taxonomic biomarkers of rUTI susceptibility in postmenopausal women and an enrichment of lactobacilli in postmenopausal women taking estrogen hormone therapy. We find robust correlations between Bifidobacterium and Lactobacillus and urinary estrogens in women without urinary tract infection (UTI) history. Functional analyses reveal distinct metabolic and antimicrobial resistance gene (ARG) signatures associated with rUTI. Importantly, we find that ARGs are enriched in the urogenital microbiomes of women with rUTI history independent of current UTI status. Our data suggest that rUTI and estrogen shape the urogenital microbiome in postmenopausal women.

Global Comparative Label-Free Yeast Proteome Analysis by LC-MS/MS After High-pH Reversed-Phase Peptide Fractionation Using Solid-Phase Extraction Cartridges.

Discovery-driven comparative proteomics employing the bottom-up strategy with label-free quantification on high-resolution mass analyzers like an Orbitrap in a hybrid instrument has the capacity to reveal unique biological processes in the context of plant metabolic engineering. However, proteins are very heterogeneous in nature with a wide range of expression levels, and overall coverage may be suboptimal regarding both the number of protein identifications and sequence coverage of the identified proteins using conventional data-dependent acquisitions without sample fractionation before online nanoflow liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). In this chapter, we detail a simple and robust method employing high-pH reversed-phase (HRP) peptide fractionation using solid-phase extraction cartridges for label-free proteomic analyses. Albeit HRP fractionation separates peptides according to their hydrophobicity like the subsequent nanoflow gradient reversed-phased LC relying on low pH mobile phase, the two methods are orthogonal. Presented here as a protocol with yeast (Saccharomyces cerevisiae) as a frequently used model organism and hydrogen peroxide to exert cellular stress and survey its impact compared to unstressed control as an example, the described workflow can be adapted to a wide range of proteome samples for applications to plant metabolic engineering research.

GC-MS/MS Profiling of Plant Metabolites.

Gas chromatography coupled to electron ionization (EI) quadrupole mass spectrometry (GC-MS) is currently one of the most developed and robust metabolomics technologies. This approach allows for simultaneous measurements of large number of chemically diverse compounds including organic acids, amino acids, sugars, sugar alcohols, aromatic amines, and fatty acids. Untargeted GC-MS profiling based on full scan data acquisition requires complicated raw data processing and sometime provides ambiguous metabolite identifications. Targeted analysis using GC-MS/MS can provide better specificity, increase sensitivity, and simplify data processing and compound identification but wider application of targeted GC-MS/MS approach in metabolomics is hampered by the lack of extensive databases of MRM transitions for non-derivatized and derivatized endogenous metabolites. The focus of this chapter is the automation of GC-MS/MS method development which makes it feasible to develop quantitative methods for several hundred metabolites and use this strategy for plant metabolomics applications.

Analysis of Grape Volatiles Using Atmospheric Pressure Ionization Gas Chromatography Mass Spectrometry-Based Metabolomics.

Analysis of volatile compounds in fruits and plants can be a challenging task as they present in a large amount with structural diversity and high aroma threshold, the information on molecular ion can be very useful for compound identification. Electron ionization gas-chromatography-mass spectrometry (EI-GC-MS) which is widely used for the analysis of plant volatiles has a certain limitation providing the limited capability to characterize novel metabolites in a complex biological matrix due to hard fragmentation level. Atmospheric pressure ionization using APGC source in combination with high-resolution time-of-flight mass spectrometry (TOF-MS) provides an excellent combination of GC with high-resolution mass spectrometry. The APGC-MS approach provides several advantages over the conventional EI and CI based GC-MS techniques in metabolomics studies due to highly reduced fragmentation, which preserves molecular ion, and accurate mass measurement by HRMS allows to deduce the elemental composition of the volatile compounds. Moreover, the use of MSE mode provides spectral similarity to EI in high-energy mode which can be used for the further confirmation of metabolite identity. We describe an APGC-MS-based untargeted metabolomics approach with a case study of grape volatile compounds and the development of a spectral library for metabolite identification.

Non-targeted Lipidomics Using a Robust and Reproducible Lipid Separation Using UPLC with Charged Surface Hybrid Technology and High-Resolution Mass Spectrometry.

Lipids play an important role in the energy storage, cellular signaling, and pathophysiology of diseases such as cancer, neurodegenerative diseases, infections, and diabetes. Due to high importance of diverse lipid classes in human health and disease, manipulating lipid abundance and composition is an important target for metabolic engineering. The extreme structural diversity of lipids in real biological samples is challenging for analytical techniques due to large difference in physicochemical properties of individual lipid species. This chapter describes lipidomic analysis of large sample sets requiring reliable and robust methodology. Rapid and robust methods facilitate the support of longitudinal studies allowing the transfer of methodology between laboratories. We describe a high-throughput reversed-phase LC-MS methodology using Ultra Performance Liquid Chromatography (UPLC®) with charged surface hybrid technology and accurate mass detection for high-throughput non-targeted lipidomics. The methodology showed excellent specificity, robustness, and reproducibility for over 100 LC-MS injections.

Comprehensive Analysis of Plant Lipids Using Sub-2-µm Particle CO2-Based Chromatography Coupled to Mass Spectrometry.

Lipids play an essential role in plants, and historically manipulating their levels and composition has been an important target for metabolic engineering. A variety of analytical techniques, many based on mass spectrometry, have been utilized for lipid profiling, but the analysis of complex lipid mixtures still poses significant analytical challenges. Recent advances in technology have revived the supercritical fluid chromatography (SFC) as a promising separation technique for lipid analysis. Utilization of sub-2-µm particle columns improves the separation efficiency and robustness of the SFC systems. The combination of SFC with sub-2-µm particle separation, commonly referred as ultra-performance convergence chromatography, has been successfully used for separation of both polar and neutral lipids. In this chapter, we present a simple method for lipid class separation using Sub-2-µm particle CO2-based chromatography coupled to mass spectrometry. The supercritical fluid chromatography methodology is flexible and can be altered to provide greater retention and separation of lipid classes or individual lipids within class.

Bioinformatics in Lipidomics: Automating Large-Scale LC-MS-Based Untargeted Lipidomics Profiling with SimLipid Software.

Liquid chromatography-mass spectrometry (LC-MS) provides one of the most popular platforms for untargeted plant lipidomics analysis (Shulaev and Chapman, Biochim Biophys Acta 1862(8):786-791, 2017; Rupasinghe and Roessner, Methods Mol Biol 1778:125-135, 2018; Welti et al., Front Biosci 12:2494-506, 2007; Shiva et al., Plant Methods 14:14, 2018). We have developed SimLipid software in order to streamline the analysis of large-volume datasets generated by LC-MS-based untargeted lipidomics methods. SimLipid contains a customizable library of lipid species; graphical user interfaces (GUIs) for visualization of raw data; the identified lipid molecules and their associated mass spectra annotated with fragment ions and parent ions; and detailed information of each identified lipid species all in a single workbench enabling users to rapidly review the results by examining the data for confident identifications of lipid molecular species. In this chapter, we present the functionality of the software and workflow for automating large-scale LC-MS-based untargeted lipidomics profiling.

Comparative Proteomics Analysis Reveals Unique Early Signaling Response of Saccharomyces cerevisiae to Oxidants with Different Mechanism of Action.

The early signaling events involved in oxidant recognition and triggering of oxidant-specific defense mechanisms to counteract oxidative stress still remain largely elusive. Our discovery driven comparative proteomics analysis revealed unique early signaling response of the yeast Saccharomyces cerevisiae on the proteome level to oxidants with a different mechanism of action as early as 3 min after treatment with four oxidants, namely H2O2, cumene hydroperoxide (CHP), and menadione and diamide, when protein abundances were compared using label-free quantification relying on a high-resolution mass analyzer (Orbitrap). We identified significant regulation of 196 proteins in response to H2O2, 569 proteins in response to CHP, 369 proteins in response to menadione and 207 proteins in response to diamide. Only 17 proteins were common across all treatments, but several more proteins were shared between two or three oxidants. Pathway analyses revealed that each oxidant triggered a unique signaling mechanism associated with cell survival and repair. Signaling pathways mostly regulated by oxidants were Ran, TOR, Rho, and eIF2. Furthermore, each oxidant regulated these pathways in a unique way indicating specificity of response to oxidants having different modes of action. We hypothesize that interplay of these signaling pathways may be important in recognizing different oxidants to trigger different downstream MAPK signaling cascades and to induce specific responses.

p63 and SOX2 Dictate Glucose Reliance and Metabolic Vulnerabilities in Squamous Cell Carcinomas.

Squamous cell carcinoma (SCC), a malignancy arising across multiple anatomical sites, is responsible for significant cancer mortality due to insufficient therapeutic options. Here, we identify exceptional glucose reliance among SCCs dictated by hyperactive GLUT1-mediated glucose influx. Mechanistically, squamous lineage transcription factors p63 and SOX2 transactivate the intronic enhancer cluster of SLC2A1. Elevated glucose influx fuels generation of NADPH and GSH, thereby heightening the anti-oxidative capacity in SCC tumors. Systemic glucose restriction by ketogenic diet and inhibiting renal glucose reabsorption with SGLT2 inhibitor precipitate intratumoral oxidative stress and tumor growth inhibition. Furthermore, reduction of blood glucose lowers blood insulin levels, which suppresses PI3K/AKT signaling in SCC cells. Clinically, we demonstrate a robust correlation between blood glucose concentration and worse survival among SCC patients. Collectively, this study identifies the exceptional glucose reliance of SCC and suggests its candidacy as a highly vulnerable cancer type to be targeted by systemic glucose restriction.

Combination of an AccQ•Tag-Ultra-Performance Liquid Chromatographic Method with Tandem Mass Spectrometry for the Analysis of Amino Acids.

Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ•Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra-performance liquid chromatography (UPLC) and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors and thoroughly tested for a variety of samples such as P. falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ•Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in Arabidopsis thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.

Metabolomics technology and bioinformatics for precision medicine.

Precision medicine is rapidly emerging as a strategy to tailor medical treatment to a small group or even individual patients based on their genetics, environment and lifestyle. Precision medicine relies heavily on developments in systems biology and omics disciplines, including metabolomics. Combination of metabolomics with sophisticated bioinformatics analysis and mathematical modeling has an extreme power to provide a metabolic snapshot of the patient over the course of disease and treatment or classifying patients into subpopulations and subgroups requiring individual medical treatment. Although a powerful approach, metabolomics have certain limitations in technology and bioinformatics. We will review various aspects of metabolomics technology and bioinformatics, from data generation, bioinformatics analysis, data fusion and mathematical modeling to data management, in the context of precision medicine.

Local and Systemic Metabolic Responses during Light-Induced Rapid Systemic Signaling.

Plants evolved multiple signaling pathways that transduce light-related signals between leaves. These are thought to improve light stress acclimation in a process termed systemic acquired acclimation. Although responses to light stress have been studied extensively in local leaves, and to a lesser degree in systemic leaves, little is known about the responses that occur in the different tissues that connect the local to the systemic leaves. These could be important in defining the specificity of the systemic response as well as in supporting the generation of different systemic signals. Here, we report that local application of light stress to one rosette leaf of bolting Arabidopsis (Arabidopsis thaliana) plants resulted in a metabolic response that encompassed local, systemic and transport tissues (stem tissues that connect the local to the systemic tissues), demonstrating a high degree of physical and metabolic continuity between different tissues throughout the plant. Our results further indicate that the response of many of the systemically altered metabolites is associated with the function of the reactive oxygen species wave and that the levels of eight different metabolites are altered in a similar manner in all tissues tested (local, systemic, and transport). These compounds could define a core metabolic signature for light stress that propagates from the local to the systemic leaves. Our findings suggest that metabolic changes occurring in cells that connect the local and systemic tissues play an important role in systemic acquired acclimation and could convey specificity to the rapid systemic response of plants to light stress.

Supercritical fluid chromatography coupled to mass spectrometry - A metabolomics perspective.

Metabolomics as a global analysis of a large number of cellular metabolites relies heavily on the new developments in separation science and technology. None of the existing analytical techniques can simultaneously separate and measure all the cellular metabolites due to complexity of cellular metabolome and, therefore, a combination of analytical techniques must be used. Currently NMR, GC-MS and LC-MS are most often used in metabolomics. Novel separation methods such as supercritical fluid chromatography (SFC), which can increase metabolome coverage while decreasing cost and analysis time, can provide alternative to other analytical techniques. As a result of major improvements in instrumentation and development of a new diverse column chemistries SFC-MS is increasingly used in a variety of biomedical applications and is becoming an attractive compliment to other major analytical platforms in metabolomics. Despite its potential and advantages, SFC-MS application in metabolomics is limited. Here we provide a brief overview of the latest developments of SFC-MS for metabolomics applications.

Rapid Accumulation of Glutathione During Light Stress in Arabidopsis.

Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. We previously reported that in response to rapid changes in light intensity (0-60 s), Arabidopsis thaliana plants mount a large-scale transcriptomic response that includes several different transcripts essential for light stress acclimation. Here, we expand our analysis of the rapid response of Arabidopsis to light stress using a metabolomics approach and identify 111 metabolites that show a significant alteration in their level during the first 90 s of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is associated with an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in these responses.

Targeting Hypoxia-Inducible Factor-1α/Pyruvate Dehydrogenase Kinase 1 Axis by Dichloroacetate Suppresses Bleomycin-induced Pulmonary Fibrosis.

Hypoxia has long been implicated in the pathogenesis of fibrotic diseases. Aberrantly activated myofibroblasts are the primary pathological driver of fibrotic progression, yet how various microenvironmental influences, such as hypoxia, contribute to their sustained activation and differentiation is poorly understood. As a defining feature of hypoxia is its impact on cellular metabolism, we sought to investigate how hypoxia-induced metabolic reprogramming affects myofibroblast differentiation and fibrotic progression, and to test the preclinical efficacy of targeting glycolytic metabolism for the treatment of pulmonary fibrosis. Bleomycin-induced pulmonary fibrotic progression was evaluated in two independent, fibroblast-specific, promoter-driven, hypoxia-inducible factor (Hif) 1A knockout mouse models and in glycolytic inhibitor, dichloroacetate-treated mice. Genetic and pharmacological approaches were used to explicate the role of metabolic reprogramming in myofibroblast differentiation. Hypoxia significantly enhanced transforming growth factor-ß-induced myofibroblast differentiation through HIF-1α, whereas overexpression of the critical HIF-1α-mediated glycolytic switch, pyruvate dehydrogenase kinase 1 (PDK1) was sufficient to activate glycolysis and potentiate myofibroblast differentiation, even in the absence of HIF-1α. Inhibition of the HIF-1α/PDK1 axis by genomic deletion of Hif1A or pharmacological inhibition of PDK1 significantly attenuated bleomycin-induced pulmonary fibrosis. Our findings suggest that HIF-1α/PDK1-mediated glycolytic reprogramming is a critical metabolic alteration that acts to promote myofibroblast differentiation and fibrotic progression, and demonstrate that targeting glycolytic metabolism may prove to be a potential therapeutic strategy for the treatment of pulmonary fibrosis.