Identification, Organic Synthesis, and Sensitive Analysis of a cis-Homosalate-Specific Exposure Biomarker.

Homosalate (HMS) is an organic UV filter used in sunscreens and personal care products. Despite its widespread use and detection in environmental matrices, little is known regarding its exposure in humans. HMS is used as a mixture of cis- and trans-isomers, and we recently revealed major differences in human toxicokinetics, indicating the need to consider these isomers separately in exposure and risk assessments. In the course of these previous investigations of human HMS toxicokinetics, we identified two trans-HMS-specific and one cis-HMS-specific biomarker candidates. However, the latter lacks sensitivity due to only low amounts excreted in urine, prompting the search for another cis-HMS-specific biomarker. Our toxicokinetic investigations revealed a total of five isomers of HMS carboxylic acid metabolites (HMS-CA). Of these, only one was specifically formed from cis-HMS (HMS-CA 5), but its full identity in terms of constitution and configuration had, so far, not been elucidated. Here, we describe the synthesis of three HMS-CA isomers, of which the isomer (1R,3S,5S)/(1S,3R,5R)-3-((2-hydroxybenzoyl)oxy)-1,5-dimethylcyclohexane-1-carboxylic acid turned out to be HMS-CA 5. Taken together with two previously synthesized HMS-CA isomers, we were able to identify the constitution and configuration of all five HMS-CA isomers observed in human metabolism. We integrated the newly identified cis-HMS-specific metabolite HMS-CA 5 into our previously published human biomonitoring LC-MS/MS method. Intra- and interday precisions had coefficients of variation below 2% and 5%, respectively, and the mean relative recovery was 96%. The limit of quantification in urine was 0.02 µg L-1, enabling the quantification of HMS-CA 5 in urine samples for at least 96 h after sunscreen application. The extended method thus enables the sensitive and separate monitoring of cis- and trans-HMS in future human biomonitoring studies for exposure and risk assessment.

LC-MS/MS-based phospholipid profiling of plant-pathogenic bacteria with tailored separation of methyl-branched species.

Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC-MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.

Toxicokinetics of homosalate in humans after dermal application: applicability of oral-route data for exposure assessment by human biomonitoring.

Homosalate (HMS) is a UV filter used in sunscreens and personal care products as a mixture of cis- and trans-isomers. Systemic absorption after sunscreen use has been demonstrated in humans, and concerns have been raised about possible endocrine activity of HMS, making a general population exposure assessment desirable. In a previous study, it was shown that the oral bioavailability of cis-HMS (cHMS) is lower than that of trans-HMS (tHMS) by a factor of 10, calling for a separate evaluation of both isomers in exposure and risk assessment. The aim of the current study is the investigation of HMS toxicokinetics after dermal exposure. Four volunteers applied a commercial sunscreen containing 10% HMS to their whole body under regular-use conditions (18-40 mg HMS (kg bw)-1). Parent HMS isomers and hydroxylated and carboxylic acid metabolites were quantified using authentic standards and isotope dilution analysis. Further metabolites were investigated semi-quantitatively. Elimination was delayed and slower compared to the oral route, and terminal elimination half-times were around 24 h. After dermal exposure, the bioavailability of cHMS was a factor of 2 lower than that of tHMS. However, metabolite ratios in relation to the respective parent isomer were very similar to the oral route, supporting the applicability of the oral-route urinary excretion fractions for dermal-route exposure assessments. Exemplary calculations of intake doses showed margins of safety between 11 and 92 (depending on the approach) after single whole-body sunscreen application. Human biomonitoring can reliably quantify oral and dermal HMS exposures and support the monitoring of exposure reduction measures.

Lipidomic and Metallomic Alteration of Caenorhabditis elegans after Acute and Chronic Manganese, Iron, and Zinc Exposure with a Link to Neurodegenerative Disorders.

Parkinson's disease (PD) progresses with the loss of dopaminergic neurons in the substantia nigra pars compacta region of the brain. The superior mechanisms and the cause of this specific localized neurodegeneration is currently unknown. However, experimental evidence indicates a link between PD progression and reactive oxygen species with imbalanced metal homeostasis. Wild-type Caenorhabditis elegans exposed to redox-active metals was used as the model organism to study cellular response to imbalanced metal homeostasis linked to neurodegenerative diseases. Using modern hyphenated techniques such as capillary electrophoresis coupled to inductively coupled plasma mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry, alterations in the lipidome and metallome were determined in vivo. In contrast to iron, most of the absorbed zinc and manganese were loosely bound. We observed changes in the phospholipid composition for acute iron and manganese exposures, as well as chronic zinc exposure. Furthermore, we focused on the mitochondrial membrane alteration due to its importance in neuronal function. However, significant changes in the inner mitochondrial membrane by determination of cardiolipin species could only be observed for acute iron exposure. These results indicate different intracellular sites of local ROS generation, depending on the redox active metal. Our study combines metallomic and lipidomic alterations as the cause and consequence to enlighten intracellular mechanisms in vivo, associated with PD progression. The mass spectrometry raw data have been deposited to the MassIVE database (https://massive.ucsd.edu) with the identifier MSV000090796 and 10.25345/C51J97C8F.

Determination of double bond positions in methyl ketones by gas chromatography-mass spectrometry using dimethyl disulfide derivatives.

RATIONALE: Methyl ketones are of interest for the application as biofuels. The fatty acid metabolism of different microbes has been rearranged to achieve a sustainable production of methyl ketones. The biofuel properties and possible further chemical modifications of these methyl ketones are influenced by their chain length, as well as their degree of saturation and the corresponding double bond position. METHODS: A method based on gas chromatography-electron ionization; mass spectrometry (GC-EI-MS) was used to determine the double bond position of methyl ketones. Derivatization using dimethyl disulfide (DMDS) and an iodine catalyst enabled the activation of the double bonds for selective fragmentation during electron ionization. The cleavage led to key fragments in the Orbitrap high-resolution mass spectrum and allowed the unequivocal localization of the double bond position of the respective monounsaturated methyl ketone. RESULTS: The double bond position of several medium chain length methyl ketones originating from the gram-negative bacterium Pseudomonas taiwanensis (P. taiwanensis) VLB120 was determined. The DMDS derivatives of methyl ketones can yield isobaric fragment ions for different possible double bond positions, which can be distinguished only using high-resolution MS. The double bond position of all methyl ketones deriving from P. taiwanensis VLB120 was the same, counting from the end of the aliphatic chain, and was determined as ω-7. CONCLUSIONS: The derivatization of medium chain length monounsaturated methyl ketones with DMDS allowed the determination of the corresponding double bond position via GC-EI-MS. High-resolution MS is needed to differentiate possible double bond positions that yield isobaric fragment ions.

Quantitative Metabolism and Urinary Elimination Kinetics of Seven Neonicotinoids and Neonicotinoid-Like Compounds in Humans.

Reverse dosimetry, i.e., calculating the dose of hazardous substances that has been taken up by humans based on measured analyte concentrations in spot urine samples, is critical for risk assessment and requires metabolic and kinetic data. We quantitatively studied the metabolism of seven major neonicotinoid and neonicotinoid-like compounds (NNIs) after single oral doses in male volunteers and determined key kinetic parameters and urinary elimination for NNIs together with their metabolites. Complete and consecutive urine samples were collected over 48 h. All samples were analyzed by tandem mass spectrometry, following liquid or gas chromatographic separation. Single- and group-specific NNI metabolites were quantified, i.e., hydroxylated and N-dealkylated NNIs and NNI-associated carboxylic acids and their glycine derivatives. Large, substance-dependent variations of key toxicokinetic parameters were observed. Mean times of concentration maxima (tmax) in urine varied between 2.0 (imidacloprid) and 25.8 h (N-desmethyl-clothianidin), whereas mean urinary elimination half-times (t1/2) were between 2.5 (acetamiprid) and 49.5 h (sulfoxaflor). Mean 48 h excretion fractions (Fue's) were between 0.03% (2-chloro-1,3-thiazole-5-carboxylic acid glycine) and 84% (clothianidin). In contrast, the interindividual differences of Fue's between the volunteers for each of the NNIs and their metabolites remained low (below a factor of 2 between the maximum and minimum derived Fue with the exception of 6-chloronicotinic acid in the acetamiprid dose study). The obtained quantitative data enabled choosing appropriate biomarkers for exposure assessment and, at the same time, for risk assessment by reverse dosimetry at current environmental exposures, i.e., comparing the calculated doses that have been taken up to currently available acceptable daily intakes of NNIs.

Identification and quantification of biosurfactants produced by the marine bacterium Alcanivorax borkumensis by hyphenated techniques.

A novel biosurfactant was discovered to be synthesized by the marine bacterium Alcanivorax borkumensis in 1992. This bacterium is abundant in marine environments affected by oil spills, where it helps to degrade alkanes and, under such conditions, produces a glycine-glucolipid biosurfactant. The biosurfactant enhances the bacterium's attachment to oil droplets and facilitates the uptake of hydrocarbons. Due to its useful properties expected, there is interest in the biotechnological production of this biosurfactant. To support this effort analytically, a method combining reversed-phase high-performance liquid chromatography (HPLC) with high-resolution mass spectrometry (HRMS) was developed, allowing the separation and identification of glycine-glucolipid congeners. Accurate mass, retention time, and characteristic fragmentation pattern were utilized for species assignment. In addition, charged-aerosol detection (CAD) was employed to enable absolute quantification without authentic standards. The methodology was used to investigate the glycine-glucolipid production by A. borkumensis SK2 using different carbon sources. Mass spectrometry allowed us to identify congeners with varying chain lengths (C6-C12) and degrees of unsaturation (0-1 double bonds) in the incorporated 3-hydroxy-alkanoic acids, some previously unknown. Quantification using CAD revealed that the titer was approximately twice as high when grown with hexadecane as with pyruvate (49 mg/L versus 22 mg/L). The main congener for both carbon sources was glc-40:0-gly, accounting for 64% with pyruvate and 85% with hexadecane as sole carbon source. With the here presented analytical suit, complex and varying glycolipids can be identified, characterized, and quantified, as here exemplarily shown for the interesting glycine-glucolipid of A. borkumensis.

Quantitation of 6-chloronicotinic acid and 2-chloro-1,3-thiazole-5-carboxylic acid and their glycine conjugates in human urine to assess neonicotinoid exposure.

Neonicotinoids and neonicotinoid-like compounds (NNIs) are widely used insecticides and their ubiquitous occurrence in the environment requires methods for exposure assessment in humans. The majority of the NNIs can be divided into 6-chloropyridinyl- and 2-chlorothiazolyl-containing compounds, suggesting the formation of the group-specific metabolites 6-chloronicotinic acid (6-CNA), 2-chloro-1,3-thiazole-5-carboxylic acid (2-CTA), and their respective glycine derivatives (6-CNA-gly, 2-CTA-gly). Here, we developed and validated an analytical method based on gas chromatography coupled to mass spectrometry (GC-MS/MS) to simultaneously analyze these four metabolites in human urine. As analytical standards for the glycine conjugates were not commercially available, we synthesized 6-CNA-gly, 2-CTA-gly, and their 13C2,15N-labeled analogs for internal standardization and quantitation by stable isotope dilution. We also ensured chromatographic separation of 6-CNA and its isomer 2-CNA. Enzymatic cleavage during sample preparation was proven unnecessary. The limits of quantitation were between 0.1 (6-CNA) and 0.4 µg/L (2-CTA-gly) and the repeatability was satisfactory (coefficient of variation was <19% over the calibration range). We analyzed 38 spot urine samples from the general population and were able to quantify 6-CNA-gly in 58% of the samples (median 0.2 µg/L). In contrast, no 6-CNA could be detected. The results are in line with well-known metabolic pathways specific in humans, that, compared to rodents, favor the formation and excretion of phase-II-metabolites (glycine derivatives) rather than phase-I metabolites (free carboxylic acids). Nevertheless, the exact source of exposure (i.e., the specific NNI) remains elusive in the general population, may even vary quantitatively between different NNIs, and also might be regional specific based on the respective use of individual NNIs. In sum, we developed a robust and sensitive analytical method for the determination of four group-specific NNI metabolites.

Tattoo Pigment Identification in Inks and Skin Biopsies of Adverse Reactions by Complementary Elemental and Molecular Bioimaging with Mass Spectral Library Matching.

Tattooing has become increasingly popular throughout society. Despite the recognized issue of adverse reactions in tattoos, regulations remain challenging with limited data available and a missing positive list. The diverse chemical properties of mostly insoluble inorganic and organic pigments pose an outstanding analytical challenge, which typically requires extensive sample preparation. Here, we present a multimodal bioimaging approach combining micro X-ray fluorescence (µXRF) and laser desorption ionization-mass spectrometry (LDI-MS) to detect the elemental and molecular composition in the same sample. The pigment structures directly absorb the laser energy, eliminating the need for matrix application. A computational data processing workflow clusters spatially resolved LDI-MS scans to merge redundant information into consensus spectra, which are then matched against new open mass spectral libraries of tattoo pigments. When applied to 13 tattoo inks and 68 skin samples from skin biopsies in adverse tattoo reactions, characteristic signal patterns of isotopes, ion adducts, and in-source fragments in LDI-MS1 scans yielded confident compound annotations across various pigment classes. Combined with µXRF, pigment annotations were achieved for all skin samples with 14 unique structures and 2 inorganic pigments, emphasizing the applicability to larger studies. The tattoo-specific spectral libraries and further information are available on the tattoo-analysis.github.io website.

Human metabolism and urinary excretion of seven neonicotinoids and neonicotinoid-like compounds after controlled oral dosages.

Few human data on exposure and toxicity are available on neonicotinoids and neonicotinoid-like compounds (NNIs), an important group of insecticides worldwide. Specifically, exposure assessment of humans by biomonitoring remains a challenge due to the lack of appropriate biomarkers. We investigated the human metabolism and metabolite excretion in urine of acetamiprid (ACE), clothianidin (CLO), flupyradifurone (FLUP), imidacloprid (IMI), sulfoxaflor (SULF), thiacloprid (THIAC) and thiamethoxam (THIAM) after single oral dosages at the currently acceptable daily intake levels of the European Food Safety Authority. Consecutive post-dose urine samples were collected up to 48 h. Suspect screening of tentative metabolites was carried out by liquid chromatography-high-resolution mass spectrometry. Screening hits were identified based on their accurate mass, isotope signal masses and ratios, product ion spectra, and excretion kinetics. We found, with the exception of SULF, extensive metabolization of NNIs to specific metabolites which were excreted next to the parent compounds. Overall, 24 metabolites were detected with signal intensities indicative of high metabolic relevance. Phase-I metabolites were predominantly derived by mono-oxidation (such as hydroxy-FLUP, -IMI, and -THIAC) and by oxidative N-desalkylation (such as N-desdifluoroethyl-FLUP and N-desmethyl-ACE, -CLO and -THIAM). IMI-olefin, obtained by dehydration of hydroxylated IMI, was identified as a major metabolite of IMI. SULF was excreted unchanged in urine. Previously reported metabolites of NNIs such as 6-chloronicotinic acid or 2-chlorothiazole-4-carboxylic acid and their glycine derivatives were detected either at low signal intensities or not at all and seem less relevant for human biomonitoring. Our highly controlled approach provides specific insight into the human metabolism of NNIs and suggests suitable biomarkers for future exposure assessment at environmentally relevant exposures.

Complementing Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging with Chromatography Data for Improved Assignment of Isobaric and Isomeric Phospholipids Utilizing Trapped Ion Mobility-Mass Spectrometry.

Lipids, such for example the multifaceted category of glycerophospholipids (GP), play a major role in many biological processes. High-resolution mass spectrometry is able to identify these highly diverse lipid species in combination with fragmentation experiments (MS/MS) on the basis of the accurate m/z and fragmentation pattern. However, for the differentiation of isomeric lipids or isobaric interferences, more elaborate separation methods are required. Especially for imaging techniques, such as matrix-assisted laser desorption/ionization (MALDI)-MS imaging, the identification is often exclusively based on the accurate m/z. Fragmentation via MS/MS increases the confidence in lipid annotation in imaging approaches. However, this is sometimes not feasible due to insufficient sensitivity and significantly prolonged analysis time. The use of a separation dimension such as trapped ion mobility spectrometry (TIMS) after ionization strengthens the confidence of the identification based on the collision cross section (CCS). Since CCS libraries are limited, a tissue-specific database was initially generated using hydrophilic interaction liquid chromatography-TIMS-MS. Using this database, the identification of isomeric lipid classes as well as isobaric interferences in a lipid class was performed using a mouse spleen sample in a workflow described in this study. Besides a CCS-based identification as an additional identification criterion for GP in general, the focus was on the distinction of the isomeric GP classes phosphatidylglycerol and bis(monoacylglycero)phosphate, as well as the differentiation of possible isobaric interferences based on the formation of adducts by MALDI-TIMS-MS imaging on a molecular level.

Human Metabolism and Urinary Excretion Kinetics of Nonylphenol in Three Volunteers after a Single Oral Dose.

Nonylphenol (NP) is an endocrine-disrupting anthropogenic chemical that is ubiquitous in the environment. Human biomonitoring data and knowledge on internal NP exposure are still sparse, and its human metabolism is largely unknown. Therefore, in this study, we investigated human metabolism and urinary excretion of NP. Three male volunteers received a single oral dose of 1 mg 13C6-labeled NP (10.6-11.7 µg/kg body weight). Consecutive full urine voids were collected for 48 h. A metabolite screening identified nine ring- and/or side chain-oxidized metabolites. We chose the most promising hits, the alkyl chain-oxidized metabolites hydroxy-NP (OH-NP) and oxo-NP, for quantitative investigation next to the parent NP. For this purpose, we newly synthesized specific n - 1-oxidized monoisomeric analytical standards. Quantification of the polyisomeric metabolites was performed via online-solid phase extraction-LC-MS/MS with stable isotope dilution using a previously published consensus method. Alkyl chain hydroxylation (OH-NP) constituted the major metabolism pathway representing 43.7 or 62.2% (depending on the mass transition used for quantification) of the NP dose excreted in urine. The urinary excretion fraction (FUE) for oxo-NP was 6.0 or 9.3%. The parent NP, quantified via an analogous isomeric 13C6-NP standard, represented 6.6%. All target analytes were excreted predominately as glucuronic acid conjugates. Excretion was rather quick, with concentration maxima in urine 2.3-3.4 h after dosing and biphasic elimination kinetics (elimination half-times first phase: 1.0-1.5 h and second phase: 5.2-6.8 h). Due to its high FUE and insusceptibility to external contamination (contrary to parent NP), OH-NP represents a robust and sensitive novel exposure biomarker for NP. The novel FUEs enable to robustly back-calculate the overall NP intakes from urinary metabolite levels in population samples for a well-informed cumulative exposure and risk assessment.

Expanding the Kendrick Mass Plot Toolbox in MZmine 2 to Enable Rapid Polymer Characterization in Liquid Chromatography-Mass Spectrometry Data Sets.

Technological advances in mass spectrometry (MS) toward more accurate and faster data acquisition result in highly informative but also more complex data sets. Especially the hyphenation of liquid chromatography (LC) and MS yields large data files containing a high amount of compound specific information. Using electrospray-ionization for compounds such as polymers enables highly sensitive detection, yet results in very complex spectra, containing multiply charged ions and adducts. Recent years have seen the development of novel or updated data mining strategies to reduce the MS spectra complexity and to ultimately simplify the data analysis workflow. Among other techniques, the Kendrick mass defect analysis, which graphically highlights compounds containing a given repeating unit, has been revitalized with applications in multiple fields of study, such as lipids and polymers. Especially for the latter, various data mining concepts have been developed, which extend regular Kendrick mass defect analysis to multiply charged ion series. The aim of this work is to collect and subsequently implement these concepts in one of the most popular open-source MS data mining software, i.e., MZmine 2, to make them rapidly available for different MS based measurement techniques and various vendor formats, with a special focus on hyphenated techniques such as LC-MS. In combination with already existing data mining modules, an example data set was processed and simplified, enabling an ever faster evaluation and polymer characterization.

Hydroperoxylated vs Dihydroxylated Lipids: Differentiation of Isomeric Cardiolipin Oxidation Products by Multidimensional Separation Techniques.

In recent years, cardiolipin (CL) oxidation products were recognized as potential markers for mitochondrial dysfunction in conjunction with age related diseases. The analysis of oxidized CL requires powerful analysis techniques due to high structural diversity. In addition, low concentrations of partly labile compounds pose a special challenge, supplemented by the occurrence of isomeric compounds, e.g., hydroperoxylated vs dihydroxylated products. Therefore, we present a hyphenated method based on liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) for separation and tandem mass spectrometry (MS/MS) for structural characterization. This enables comprehensive analysis of an artificially oxidized CL extract of bovine heart. Isomeric oxidation products could be differentiated by mobility-resolved MS/MS fragmentation experiments. Our developed method could help to better understand the physiological role of oxidized CL.

Exploiting the Natural Diversity of RhlA Acyltransferases for the Synthesis of the Rhamnolipid Precursor 3-(3-Hydroxyalkanoyloxy)Alkanoic Acid.

While rhamnolipids of the Pseudomonas aeruginosa type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new rhlA genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in Betaproteobacteria and Gammaproteobacteria A likely horizontal gene transfer event into Actinobacteria is the only identified exception. The phylogeny of the RhlA homologs from Pseudomonas and Burkholderia species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the Enterobacterales do not follow the organisms' phylogeny but form their own branch. Furthermore, in many Enterobacterales and Halomonas from the Oceanospirillales, an isolated rhlA homolog can be found in the genome. The RhlAs from Pseudomonas aeruginosa PA01, Pseudomonas fluorescens LMG 05825, Pantoea ananatis LMG 20103, Burkholderia plantarii PG1, Burkholderia ambifaria LMG 19182, Halomonas sp. strain R57-5, Dickeya dadantii Ech586, and Serratia plymuthica PRI-2C were expressed in Escherichia coli and tested for HAA production. Indeed, except for the Serratia RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C10, C12, and C14 fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity.IMPORTANCE The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.

Analysis of artificially oxidized cardiolipins and monolyso-cardiolipins via liquid chromatography/high-resolution mass spectrometry and Kendrick mass defect plots after hydrophilic interaction liquid chromatography based sample preparation.

RATIONALE: Cardiolipins (CL) are a special lipid class which plays a main role in energy metabolism in mitochondria and is involved in apoptosis. In contrast to other glycerophospholipids, they contain four fatty acyl residues which results in a high structural diversity. Oxidation, for example by reactive oxygen species, or lyso forms such as monolyso-CL (MLCL), increases this diversity. Mass spectrometric analysis and computational identification of CL, MLCL and their oxidation products is therefore a challenging task. METHODS: In order to distinguish CL, MLCL and their oxidation products, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. A hydrophilic interaction liquid chromatography (HILIC)-based solid-phase extraction (SPE) clean-up approach was developed for CL enrichment. Graphical analysis of CL, MLCL and their oxidation products was carried out by a three-dimensional Kendrick mass defect (3D-KMD) plot module, as well as a refined lipid search module of the open-source metabolomics data mining software MZmine 2. RESULTS: The HILIC-based SPE clean-up enabled complete separation of polar and nonpolar lipid classes. A yeast (Saccharomyces cerevisiae) lipid extract, which was artificially oxidized by means of the Fenton reaction, was analyzed by the developed LC/MS/MS method. CL species with differences in chain length and degree of unsaturation have been separated by high-performance liquid chromatography (HPLC). In total 66 CL, MLCL and oxidized species have been identified utilizing 3D-KMD plots in combination with database matching using MZmine 2. For further characterization of annotated species, MS/MS experiments have been utilized. CONCLUSIONS: 3D-KMD plots capturing chromatographic and high-resolution mass spectrometry data have been successfully used for graphical identification of CL, MLCL as well as their oxidized species. Therefore, we chose multiple KMD bases such as hydrogen and oxygen to visualize the degree of unsaturation and oxidation capturing chromatographic data by means of a color-coded paint scale as the third dimension. In combination with database matching, the analysis of low concentrated lipid species in complex samples has been significantly improved.

Identification and structural characterization of lipid A from Escherichia coli, Pseudomonas putida and Pseudomonas taiwanensis using liquid chromatography coupled to high-resolution tandem mass spectrometry.

RATIONALE: Lipid A is a part of the lipopolysaccharide layer, which is a main component of the outer membrane from Gram-negative bacteria. It can be sensed by mammalians to identify the presence of Gram-negative bacteria in their tissues and plays a key role in the pathogenesis of bacterial infections. Lipid A is also used as an adjuvant in human vaccines, emphasizing the importance of its structural analysis. METHODS: In order to distinguish and characterize various lipid A species, a liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) method was developed. Isolation of lipid A from different bacteria was carried out using a modified Bligh and Dyer extraction following a mild acid hydrolysis. Chromatography was performed using a bifunctional reversed-phase-based stationary phase. High-resolution MS using negative electrospray ionization was applied and MS/MS experiments utilizing high-energy collisional dissociation generated diagnostic product ions, which allowed the assignment of the side chains to distinct positions of the lipid A backbone. RESULTS: The method was applied to lipid A isolations of Escherichia coli (E. coli), Pseudomonas putida (P. putida) and Pseudomonas taiwanensis (P. taiwanensis). Various lipid A species were identified by their accurate masses and their structures were characterized using MS/MS experiments. Previously described lipid A structures from E. coli were identified and their structures confirmed by MS/MS. For the biotechnologically relevant strains P. putida and P. taiwanensis, we confirmed species by MS/MS, which have previously only been analyzed using MS. In addition, several lipid A species were discovered that have not been previously described in the literature. CONCLUSIONS: The combination of LC and MS/MS enabled the selective and sensitive identification and structural characterization of various lipid A species from Gram-negative bacteria. These species varied in their substituted side chains, speaking of fatty acids and phosphate groups. Characteristic product ions facilitated the assignment of side chains to distinct positions of the lipid A backbone.

Double bond localization in unsaturated rhamnolipid precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids by liquid chromatography-mass spectrometry applying online Paternò-Büchi reaction.

Lipids are biomolecules with a broad variety of chemical structures, which renders them essential not only for various biological functions but also interestingly for biotechnological applications. Rhamnolipids are microbial glycolipids with surface-active properties and are widely used biosurfactants. They are composed of one or two L-rhamnoses and up to three hydroxy fatty acids. Their biosynthetic precursors are 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs). The latter are also present in cell supernatants as complex mixtures and are extensively studied for their potential to replace synthetically derived surfactants. The carbon chain lengths of HAAs determine their physical properties, such as their abilities to foam and emulsify, and their critical micelle concentration. Despite growing biotechnological interest, methods for structural elucidation are limited and often rely on hydrolysis and analysis of free hydroxy fatty acids losing the connectivity information. Therefore, a high-performance liquid chromatography-mass spectrometry method was developed for comprehensive structural characterization of intact HAAs. Information is provided on chain length and number of double bonds in each hydroxy fatty acid and their linkage by tandem mass spectrometry (MS/MS). Post-column photochemical derivatization by online Paternὸ-Büchi reaction and MS/MS fragmentation experiments generated diagnostic fragments allowing structural characterization down to the double bond position level. Furthermore, the presented experiments demonstrate a powerful approach for structure elucidation of complex lipids by tailored fragmentation.

Importance of oxidation products in coumarin-mediated Fe(hydr)oxide mineral dissolution.

Due to the low iron solubility in alkaline soils, plants have evolved different iron acquisition strategies, which are either based on ferric iron reduction (strategy I) or complexation by phytosiderophores (strategy II). Recently, a prominent role of coumarins for iron acquisition has been discovered, but details of the respective mechanism remain unclear. Since coumarins may act as iron-binding ligands but also as reductants, various reaction sequences are possible, resulting in different iron species and oxidized coumarins. In this context, it is often overlooked that oxidized coumarins are not just byproducts of iron(III) reduction, but may be actively involved in further steps of iron mobilization. In order to verify this active role of oxidized coumarins in Fe(hydr)oxide dissolution, we complemented iron dissolution data with data of single coumarins (esculetin, scopoletin, fraxetin) and their oxidation products, as a function of time, pH, and mineral (goethite, lepidocrocite). Our results demonstrate that there are four different routes for coumarin oxidation, leading to quinones, dimers, hydroxylated coumarins, demethylated coumarins, and combinations of these. The time-dependent species pattern differs with respect to mineral, pH, and coumarin molecule. Oxidized coumarins are often more reactive than the original coumarins, explaining unexpected iron mobilization by scopoletin, which is demethylated to esculetin. Also oxidative hydroxylation and dimerization increase the number of phenolic groups and yield new chelating properties. Several iron-species are identified for the three coumarins. Since oxidation reactions are initiated directly at mineral surfaces, they are often very effective-but this does not always result in more iron mobilization.

LIPG-promoted lipid storage mediates adaptation to oxidative stress in breast cancer.

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression.