Comprehensive profiling of <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones produced by <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> using liquid chromatography coupled to hybrid quadrupole–linear ion trap mass spectrometry

A method for the comprehensive profiling of the N-acylhomoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to hybrid quadrupole–linear ion trap (QqQLIT) mass spectrometry. Information-dependent acquisition (IDA), using triggered combinations of triple-quadrupole and linear ion trap modes in the same LC-MS/MS run, was used to simultaneously screen, quantify and identify multiple AHLs in a single sample. This MS method uses common AHL fragment ions attributed to the homoserine moiety and the 3-oxo-, 3-hydroxy- or unsubstituted acyl side chains, to identify unknown AHLs in cell-free culture supernatants in an unbiased manner. This LC-MS technique was applied to determine the relative molar ratios of AHLs produced by Yersinia pseudotuberculosis and the consequences of inactivating by mutation either or both of the AHL synthase genes (ypsI and ytbI) on AHL profile and concentration. The Y. pseudotuberculosis wild type but not the ypsI ytbI double mutant produced at least 24 different AHLs with acyl chains ranging from C4 to C15 with or without 3-oxo or 3-hydroxy substituents. YtbI, in contrast to YpsI, could direct the synthesis of all of the AHLs identified. The most abundant and hence most biologically relevant Y. pseudotuberculosis AHLs were found to be the 3-oxo-substituted C6, C7 and C8 AHLs and the unsubstituted C6 and C8 compounds. The LC-QqQLIT methodology is broadly applicable to quorum-sensing signal molecule analysis and can provide comprehensive AHL profiles and concentrations from a single sample and simultaneously collect confirmatory spectra for each AHL identified. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Catharine A. Ortori and Steve Atkinson made an equal contribution to the paper.     

Uptake, degradation and chiral discrimination of N-acyl-D/L-homoserine lactones by barley (Hordeum vulgare) and yam bean (Pachyrhizus erosus) plants

Bacterial intraspecies and interspecies communication in the rhizosphere is mediated by diffusible signal molecules. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as autoinducers in the quorum sensing response. While bacterial signalling is well described, the fate of AHLs in contact with plants is much less known. Thus, adsorption, uptake and translocation of N-hexanoyl- (C6-HSL), N-octanoyl- (C8-HSL) and N-decanoyl-homoserine lactone (C10-HSL) were studied in axenic systems with barley (Hordeum vulgare L.) and the legume yam bean (Pachyrhizus erosus (L.) Urban) as model plants using ultra-performance liquid chromatography (UPLC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tritium-labelled AHLs. Decreases in AHL concentration due to abiotic adsorption or degradation were tolerable under the experimental conditions. The presence of plants enhanced AHL decline in media depending on the compounds' lipophilicity, whereby the legume caused stronger AHL decrease than barley. All tested AHLs were traceable in root extracts of both plants. While all AHLs except C10-HSL were detectable in barley shoots, only C6-HSL was found in shoots of yam bean. Furthermore, tritium-labelled AHLs were used to determine short-term uptake kinetics. Chiral separation by GC-MS revealed that both plants discriminated D-AHL stereoisomers to different extents. These results indicate substantial differences in uptake and degradation of different AHLs in the plants tested.     

Detection of bacterial quorum sensing <Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactones in clinical samples

Bacteria communicate among themselves using certain chemical signaling molecules. These signaling molecules generally are N-acyl homoserine lactones (AHLs) in Gram-negative bacteria and oligopeptides in Gram-positive bacteria. In addition, both Gram-positive and Gram-negative bacteria produce a family of signaling molecules known as autoinducer-2 that they employ for their communications. Bacteria coordinate their behavior by releasing and responding to the chemical signaling molecules present in proportion to their population density. This phenomenon is known as quorum sensing. The role of bacteria in the pathogenesis of several diseases, including gastrointestinal (GI) disorders, is well established. Moreover, rather recently bacterial quorum sensing has been implicated in the onset of bacterial pathogenicity. Thus, we hypothesized that the signaling molecules involved in bacterial communication may serve as potential biomarkers for the diagnosis and management of several bacteria-related diseases. For that, we previously developed a method based on genetically engineered whole-cell sensing systems for the rapid, sensitive, cost-effective and quantitative detection of AHLs in biological samples, such as saliva and stool, from both healthy and diseased individuals with GI disorders. Although various analytical methods, based on physical-chemical techniques and bacterial whole-cell biosensors, have been developed for the detection of AHLs in the supernatants of bacterial cultures, only a few of them have been applied to AHL monitoring in real samples. In this paper, we report work performed in our laboratory and review that from others that describes the detection of AHLs in biological, clinical samples, and report some of our recent experimental results.     

At-line coupling of UPLC to chip-electrospray-FTICR-MS

Since highly sensitive on-line coupling of UPLC with FTICR-MS is technically infeasible due to their different scan rates, at-line coupling of these techniques was developed for rapid analysis. To enable cutting of one peak of the chromatogram into one fraction, several conditions and relationships were investigated, e.g. the optimum volume of the inserted delay loop, the relationship between retention time, loop outlet drop speed, individual drop volume versus mobile phase composition under constant speed, and linear solvent strength gradient elution modes. Good and reproducible results were achieved applying UPLC as an efficient separation and fast fractionation tool before the FTICR-MS measurements. A chip-based nanoelectrospray ionization system was employed which was perfectly suited to handling the small-volume fractions and was thus chosen for the at-line coupling. The method was initially applied to spiked extracts of cell-free bacterial culture supernatants in which bacterial signalling compounds, namely N-acyl homoserine lactones (AHL), were detected. Good reproducibility and high recovery was observed. Afterwards, a culture supernatant of Erwinia sp. JX3.2, a putative AHL producer, was investigated and N-hexanoyl-homoserine lactone was determined as a possible signalling molecule. More reliable assignments were achieved by use of at-line coupling of UPLC and FTICR-MS compared with off-line measurements. Xiaojing Li and Agnes Fekete have contributed equally to this publication.     

Deuterium-labelled N-acyl-L-homoserine lactones (AHLs)--inter-kingdom signalling molecules--synthesis, structural studies, and interactions with model lipid membranes

N-Acyl-l-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-l-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum’s acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein.     

Perceiving the chemical language of Gram-negative bacteria: listening by high-resolution mass spectrometry

Gram-negative bacteria use N-acylhomoserine lactones (AHLs) as their command language to coordinate population behavior during invasion and colonization of higher organisms. Although many different bacterial bioreporters are available for AHLs monitoring, in which a phenotypic response, e.g. bioluminescence, violacin production, and β-galactosidase activity, is exploited, mass spectrometry (MS) is the most versatile detector for rapid analysis of AHLs in complex microbial samples, with or without prior separation steps. In this paper we critically review recent advances in the application of high-resolution MS to analysis of the quorum sensing (QS) signaling molecules used by Gram-negative bacteria, with much emphasis on AHLs. A critical review of the use of bioreporters in the study of AHLs is followed by a short methodological survey of the capabilities of high-resolution mass spectrometry (HRMS), including Fourier-transform ion cyclotron resonance (FTICR) MS and quadrupole time-of-flight (qTOF) MS. Use of infusion electrospray ultrahigh-resolution FTICR MS (12 Tesla) enables accurate mass measurements for determination of the elemental formulas of AHLs in Acidovorax sp. N35 and Burkholderia ubonensis AB030584. Results obtained by coupling liquid chromatography with a hybrid quadrupole linear ion trap-FTICR mass spectrometer (LC–LTQ-FTICRMS, 7-T) for characterization of acylated homoserine lactones in the human pathogen Pseudomonas aeruginosa are presented. UPLC–ESI-qTOF MS has also proved to be suitable for identification of 3O-C₁₀HSL in Pseudomonas putida IsoF cell culture supernatant. Aspects of sample preparation and the avoidance of analytical pitfalls are also emphasized.

Use of solid-phase extraction to enable enhanced detection of acyl homoserine lactones (AHLs) in environmental samples

A challenge for understanding the role of bacterial cell–cell signalling in the environment is the detection of those signals, which are often present in low (nmol L⁻¹) concentrations. We describe here a simple purification method, solid-phase extraction (SPE), for increasing the sensitivity of detection for one such group of signals, acyl homoserine lactones (AHLs), in environmental samples. Spiking of dried marine sponge tissue (Stylinos sp.) with AHLs resulted in detection down to 0.01 ppm for 3-oxo-hexanoyl homoserine lactone (3-oxo C₆-HSL) and 1 ppm for hexanoyl homoserine lactone (C₆-HSL). Compared with liquid extraction methods use of SPE resulted in twofold and tenfold improvements in sensitivity, respectively.     

Synthesis of new 1,4- and 1,5-disubstituted N-ethyl acetate and N-α-butyro-γ-lactone alkylimidazole derivatives as N-acylhomoserine lactone analogs

New alkylimidazoles functionalized with a homoserine lactone or an alkyloxycarbonyl moiety have been synthesized as N-acylhomoserine lactones (AHLs) analogs. The 1,4-disubstituted imidazole derivatives were prepared by alkylation of 4(5)-alkylimidazoles with α-bromo-γ-butyrolactone or ethyl α-bromoacetate. An alternative route was preferred for the synthesis of their 1,5-disubstituted counterparts based on the use of a N₁-protected alkylimidazole, its alkylation to an N₃-imidazolyl-α-acetate and deprotection to the desired 1,5-disubstituted esters and subsequent alkylation of the acetate moiety with cyclic ethylene sulfate followed by acid-catalyzed cyclization. The ability to modulate bacterial quorum sensing of all new compounds was compared to that of previously reported AHL analogs in which the amide bond is replaced by a heterocyclic group.     

Identification of unsaturated N-acylhomoserine lactones in bacterial isolates of Rhodobacter sphaeroides by liquid chromatography coupled to electrospray ionization-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry

The identification of two unsaturated N‐acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)‐Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Besides the confirmation of the signaling molecule already described in the literature, i.e. (Z)‐N‐tetradec‐7‐enoyl‐homoserine lactone (C_14:1‐HSL), we have discovered the occurrence, at low, yet significant levels, of another monounsaturated compound, C_12:1‐HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high‐resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion into bromohydrins. Collision‐induced dissociation (CID) spectra were then collected on the LTQ mass analyzer. A careful comparison of tandem MS spectra of monounsaturated (i.e., C_12:1‐HSL and C_14:1‐HSL) and saturated AHLs (i.e. C₁₂‐HSL and C₁₄‐HSL) led to the emphasis of two series of product ions, exhibiting 14 Da spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e. CH₂=CH(CH₂)_nH). In particular, the series located at the higher end of the explored m/z range (>200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of diketopiperazines of Burkholderia cepacia CF-66 by gas chromatography–mass spectrometry

Bacteria communicate with each other by a process termed “quorum sensing” (QS), and diffusible, low-molecular-weight chemicals, called signal molecules, are used as the communication languages. In cell-free Burkholderia cepacia CF-66 culture supernatants, five compounds suspected of being signal molecules were identified. The gene (cepI) related with AHLs synthesis were not detected by polymerase chain reaction (PCR) using specific primers. Gas chromatography–mass spectrometry (GC–MS) revealed that these compounds were not AHLs but the diketopiperazines (DKPs) cyclo(Pro–Phe), cyclo(Pro–Tyr), cyclo(Ala–Val), cyclo(Pro–Leu), and cyclo(Pro–Val), all of which were both d and l-type. Four kinds of DKPs had been isolated from other Gram-negative bacteria, but the other was a novel kind discovered in CF-66, and l-cyclo (Pro–Phe) was quantified by GC–MS. It was found that exogenous DKPs had a negative effect on the candidacidal activity of the culture supernatant extracts.     

(Z)-N-(4-Decenoyl)homoserine lactone, a new quorum-sensing molecule, produced by endobacteria associated with Mortierella alpina A-178

N-Acylhomoserine lactones (AHLs) are the conserved quorum-sensing signal molecules in Gram-negative bacteria. (Z)-N-(4-Decenoyl)homoserine lactone (1), a new AHL, was isolated from the culture broth of the fungus Mortierella alpina A-178 harboring bacterial endosymbionts, called endobacteria. The structure and absolute configuration of 1 were elucidated by EI-MS, chemical synthesis, and chiral GC analysis. The compound induced the expression of a QS-regulated reporter gene in Agrobacterium tumefaciens NTL4, although its activity was lower than that of N-decanoylhomoserine lactone (6).     

Identification of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone by reversed‐phase high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A reversed‐phase high‐performance liquid chromatography/electrospray tandem mass spectrometry method was developed for the characterization of hydroxyl radical oxidation products of N‐hexanoyl‐homoserine lactone (C6‐HSL), a member of the N‐acylhomoserine lactone (AHL) class of microbial quorum‐sensing signaling molecules identified in many Gram‐negative strains of bacteria. Six products were identified: four with molecular weight (MW) of 213 and two with MW of 260. The characteristic product ions formed through collision‐induced dissociation (CID) provided diagnostic structural information. One of the photolysis products was determined to be N‐(3‐oxohexanoyl)homoserine lactone (3OC6‐HSL), a highly active quorum‐sensing signal, by comparison with a reference standard. Three structural isomers with the same mass as 3OC6‐HSL were identified as acyl side chain oxidized C6‐HSL (keto/enol functionalized) by accurate mass measurement and the structures of these products were proposed from CID spectral interpretation. Two structural isomers formed from concurrent oxidation and nitration of C6‐HSL were also observed and their structures were postulated based on CID spectra. In addition to the six hydroxyl radical oxidation products formed from the C6‐HSL precursor, five additional compounds generated from combined oxidation and lactonolysis of C6‐HSL were identified and structures were postulated. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and Quantification of <Emphasis Type="Italic">N</Emphasis>-Acyl Homoserine Lactones Involved in Bacterial Communication by Small-Scale Synthesis of Internal Standards and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa.

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Biosynthesis of Cd-bound phytochelatins by <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and their speciation by size-exclusion chromatography and ion-pair chromatography coupled to ICP–MS

Cd-bound phytochelatins (Cd–PCs) have been synthesised by incubation of Phaeodactylum tricornutum cell cultures with Cd and purified by size-exclusion chromatography–UV–Vis. These complexes, which were identified in previous work, have now been used as model substances to develop and optimise ion-pair chromatography (IPC) coupled to inductively coupled plasma–mass spectrometry (ICP–MS) for analysis of Cd–PCs. Subsequent analysis of samples taken from Silene vulgaris plants cultivated under heavy metal stress conditions revealed Cd signals but no Cd–PC signals. By use of isotopically enriched ¹¹⁶Cd–PCs the sample preparation steps were verified to determine the stability of the analytes. We observed species transformation between Cd–PCs and other unidentified Cd complexes. Consequently, the kinetic and thermodynamic lability of Cd–PCs are decisive factors in their detection.     

Analytical pitfalls in hair testing

This review focuses on possible pitfalls in hair testing procedures. Knowledge of such pitfalls is useful when developing and validating methods, since it can be used to avoid wrong results as well as wrong interpretations of correct results. In recent years, remarkable advances in sensitive and specific analytical techniques have enabled the analysis of drugs in alternative biological specimens such as hair. Modern analytical procedures for the determination of drugs in hair specimens—mainly by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS)—are reviewed and critically discussed. Many tables containing information related to this topic are provided.     

Fungicidal lipid-transfer peptide from <Emphasis Type="Italic">Daucus carota sativa</Emphasis> seeds

A non-specific lipid-transfer peptide (nsLTP) with fungicidal activity was isolated from Daucus carota sativa carrot seeds. Peptides were purified by a method including aqueous extraction, anion-exchange chromatography over CM-TSK-650M, and HPLC over a column of 250/8/4 Protein@Peptide C₁₈ using an acetonitrile gradient. The molecular weight of the peptide was determined as 9624 Da by mass spectrometry. The peptide was found to have fungicidal activity against the pathogenic fungus Verticillium dahliae. The partial N-terminal sequence, which was highly homologous to the N-terminal sequences of lipid-transfer peptides from seeds of rice, tobacco, and maize, was determined using Edman automated sequencing. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 371–373, July–August, 2007.     

Analysis of N-acyl-L-homoserine lactones produced by Burkholderia cepacia with partial filling micellar electrokinetic chromatography--electrospray ionization-ion trap mass spectrometry

A method for the analysis of N-acyl-L-homoserine lactones (AHLs) with micellar electrokinetic chromatography coupled to electrospray ionization-ion trap mass spectrometry, combining the flexibility of capillary electrophoresis with the unmatched structural information provided by mass spectrometry is presented. Different surfactants were evaluated, with sodium dodecyl sulfate (SDS) yielding the best results considering sensitivity and flexibility. We examined the interaction of AHLs with the SDS micelles at different analysis conditions and applied the optimized method to the analysis of a real bacterial sample. Two AHLs from Burkholderia cepacia colonizing the rhizosphere of traditional Indian rice cultivars could be unambiguously determined in an ethyl acetate extract with high resolution flexibility.     

Determination of jasmonic acid in <Emphasis Type="Italic">Lemna minor</Emphasis> (L.) by liquid chromatography with fluorescence detection

A new method is described for the determination of endogenous jasmonic acid (JA) in Lemna minor plant extracts using liquid chromatography (LC) with fluorescence detection. Plant tissues were extracted and derivatised using 9-anthryldiazomethane (ADAM reagent) prepared in situ. Accuracy and precision were improved by using the internal standard dihydrojasmonic acid (dh-JA) for the correction of JA losses during sample preparation steps. Liquid chromatography–mass spectrometry (LC/MS) analysis of ADAM derivatives of JA and dh-JA confirmed that a single molecule of JA and dh-JA was coupled with one molecule of reagent. Derivatives of JA and dh-JA were separated with gradient elution on a C₁₈ reversed-phase column using acetonitrile/water as a mobile phase and detected by a fluorescence detector at excitation and emission wavelengths of 254 and 412 nm, respectively. The detection limits of JA and dh-JA were 2.9 ng mL⁻¹ and 3.7 ng mL⁻¹ per 50-μL injection. The method is reproducible and selective and yields single peaks for each compound regardless of isomer. The specificity and accuracy of the proposed LC/FD method was confirmed by liquid chromatography–TurboIon Spray tandem mass spectrometric (LC/MS/MS) analysis of free JA in Lemna minor samples under multiple reaction monitoring conditions.     

Environmental analysis of fluorinated alkyl substances by liquid chromatography–(tandem) mass spectrometry: a review

Fluorinated alkyl substances (FASs) are widely distributed contaminants that have been found in many environmental, human and biological samples throughout the world. Perfluorochemicals are used in many industry and consumer products, such as polymers and surfactants, because they have unique and useful properties (they are stable, chemically inert and generally unreactive). However, these compounds have also been found to be toxic, persistent and bioaccumulative. In recent years various analytical methods have been developed for the analysis of FASs in environmental samples. Most of these methods are based on liquid chromatography coupled to mass spectrometry (LC–MS) or tandem mass spectrometry (LC–MS/MS), since this is considered to be the technique of choice. This article reviews the various LC–(tandem)MS methods described so far for the analysis of FASs in water, sediment, sludge and biota samples. It discusses the main experimental conditions used for sample pretreatment and for analysis as well as the most relevant problems encountered and the limits of detection achieved.     

Bioactive metabolites of the marine actinobacterium <Emphasis Type="Italic">Streptomyces</Emphasis> sp. KMM 7210

New 3-(4-hydroxybenzyl)piperazine-2,5-dione, together with the known N-[2-(4-hydroxy-phenyl)ethyl]acetamide (N-acetyltyramine), was isolated for the first time from the marine actinobacterium Streptomyces sp. The chemical structures of these compounds were determined by NMR spectroscopy and mass spectrometry. The cytotoxic activities of the compounds were estimated from their effects on sperm and eggs of the sea urchin Strongylocentrotus intermedius. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 807–809, April, 2007.