a-method-for-measuring-metabolism-sorted-subpopulations-complex-cell

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Karolinska Institutet

This work focuses on the influence of the selected LC-HRMS platform on the final annotated compounds in non-targeted metabolomics. Two platforms that differed in columns, mobile phases, gradients, chromatographs, mass spectrometers (Orbitrap [Platform#1] and Q-TOF [Platform#2]), data processing and marker selection protocols were compared. A total of 42 wines samples from three different protected denomination of origin (PDO) were analyzed. At the feature level, good (O)PLS-DA models were obtained for both platforms (Q(2)[Platform#1]=0.89, 0.83 and 0.72; Q(2)[Platform#2]=0.86, 0.86 and 0.77 for Penedes, Ribera del Duero and Rioja wines respectively) with 100% correctly classified samples in all cases. At the annotated metabolite level, platforms proposed 9 and 8 annotated metabolites respectively which were identified by matching standards or the MS/MS spectra of the compounds. At this stage, there was no coincidence among platforms regarding the suggested metabolites. When screened on the raw data, 6 and 5 of these compounds were detected on the other platform with a similar trend. Some of the detected metabolites showed complimentary information when integrated on biological pathways. Through the use of some examples at the annotated metabolite level, possible explanations of this initial divergence on the results are presented. This work shows the complications that may arise on the comparison of non-targeted metabolomics platforms even when metabolite focused approaches are used in the identification.

Told through the wine: A liquid chromatography-mass spectrometry interplatform comparison reveals the influence of the global approach on the final annotated metabolites in non-targeted metabolomics.

We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for analyzing bisphenol A (BPA) and its halogenated derivatives. Since only tetrachlorobisphenol A and tetrabromobisphenol A (TBBPA) are commercially available, mono-, di- and trichlorobisphenol A were synthesized and purified in order to be used as analytical standards. This family of compounds was studied using electrospray ionization and an ion trap mass analyzer in order to characterize the new compounds and to propose fragmentation pathways. Multi-stage mass spectrometry was used to confirm the genealogical relationship between the ions. Some product ions were traced from MS/MS to MS(4) and the labelled compounds BPA-d(16) and TBBPA-(13)C(12) were used to assign some product ion structures. In general, the deprotonated molecule [M--H](-) loses a methyl and/or a halogen group during both MS/MS and MS(3), while the neutral loss of CO was also observed in MS(3) spectra. We selected the most intense and characteristic MS/MS transitions for LC/MS/MS analysis. LC separation was performed in a reversed-phase column; methanol/water (no additives) was used as the mobile phase in gradient elution mode; and BPA-d(16) was chosen as the internal standard. Solid-phase extraction (SPE) was used to pre-concentrate and to clean up water samples. The SPE LC/MS/MS method allows BPA and its halogenated derivatives to be detected at a few parts-per-billion (ppb) in surface water.

Liquid chromatography/multi-stage mass spectrometry of bisphenol A and its halogenated derivatives.

Conditions were established for the separation and analysis of bisphenol A, bisphenol F, and their diglycidyl ethers by micellar electrokinetic capillary chromatography (MECC). Good resolution was obtained for all compounds, although in order to achieve the separation of ortho-ortho, ortho-para, and para-para isomers of bisphenol F diglycidyl ether (BFDGE), BFDGE x 2H(2)O and BFDGE x 2HCl, it was necessary to use a 25 microm id fused silica capillary. To increase sensitivity, a field-amplified sample injection (FASI)-MECC method was developed using 10 mM SDS solution as injection matrix and a 75 microm id fused silica capillary. Instrumental quality parameters such as LODs (<55 microg/L with standards), linearity (r(2)>0.999), and run-to-run and day-to-day precisions (RSD values lower than 12.5%) were determined. Finally, the suitability of the FASI-MECC method for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers in canned soft drinks was evaluated. Quantitation was performed by matrix-matched calibration using a plastic-bottled isotonic drink as matrix. The results showed that FASI-MECC is an economic method for the screening and quantitation of these kinds of compounds in soft drink beverages, with no loss of reproducibility, and effective at concentrations lower than the specific migration level values established by the European Union.

Field-amplified sample injection-micellar electrokinetic capillary chromatography for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers and derivatives in canned soft drinks.

A fast method of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed for the analysis of eleven UV ink photoinitiators in packaged food. Chromatographic separation was achieved in a pentafluorophenylpropyl (PFPP) column at 5°C and acetonitrile:25 mM formic acid-ammonium formate (pH 2.7) in gradient elution. To reduce sample treatment, a QuEChERS (quick, easy, cheap, effective, rugged and safe) method for the extraction and clean-up of UV photoinitiators in packaged foods was evaluated. Triple quadrupole working in H-SRM on Q1 mode was used for both quantitation and confirmation purposes and the most intense and selective transitions were chosen. Quality parameters of the developed QuEChERS LC-MS/MS method were established and applied for the analysis of photoinitiators in food packaged at ng kg(-1) levels.

Analysis of UV ink photoinitiators in packaged food by fast liquid chromatography at sub-ambient temperature coupled to tandem mass spectrometry.

Environmental pollution by pentachlorophenol (PCP) is a critical concern worldwide and fungal bioremediation constitutes an elegant and environment-friendly solution. Mucorales from the Zygomycota phylum are often observed to be competitive in field conditions and Mucor plumbeus, in particular, can efficiently deplete PCP from media. The pathway for PCP degradation used by this fungus has not been investigated. In this study, PCP-derived metabolites were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, including tetra- and tri-chlorohydroquinones and phase II-conjugated metabolites. Amongst the latter are the previously reported glucose, sulfate and ribose conjugates, and identified for the first time in fungi sulfate-glucose conjugates. A PCP transformation pathway for M. plumbeus is proposed, which excludes the involvement of cytochrome P-450 and extracellular ligninolytic enzymes.

Degradation pathway of pentachlorophenol by Mucor plumbeus involves phase II conjugation and oxidation-reduction reactions.

There is an increasing need for applications in food and environmental areas able to cope with a large number of analytes in very complex matrices. The new analytical procedures demand sensitivity, robustness and high resolution within an acceptable analysis time. The purpose of this review is to describe new trends based on fast liquid chromatography applied to the food and environmental analysis. It includes different column technologies, such as monolithic, sub-2 μm, porous shell, as well as different stationary phases such as reversed phase (C8 and C18), hydrophilic interaction liquid chromatography (HILIC) and fluorinated columns. Additionally, recent sample extraction and clean-up methodologies applied to reduce sample manipulation and total analysis time in food and environmental analysis--QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), on line solid phase extraction coupled to ultrahigh pressure liquid chromatography (on line SPE-UHPLC), turbulent flow chromatography (TFC) and molecularly imprinted polymers (MIPs), were also addressed. The advantages and drawbacks of these methodologies applied to the fast and sensitive analyses of food and environmental samples are going to be discussed.

New trends in fast liquid chromatography for food and environmental analysis.

Atmospheric pressure photoionization (APPI) was evaluated for the analysis of fullerenes. An important response improvement was found when using toluene mediated APPI in negative mode if compared with other atmospheric pressure ionization (API) sources (electrospray and atmospheric pressure chemical ionization). Fullerene APPI negative mass spectra were dominated by the isotopic cluster of the molecular ion, although isotopic patterns for M+1, M+2, and M+3 ions showed higher than expected relative abundances. These discrepancies are explained by the presence of two isobaric ions, one due to (13)C and the other due to the addition of hydrogen to a double bond of the fullerene structure. Triple quadrupole tandem mass spectrometry, ultrahigh resolution mass spectrometry, and accurate mass measurements were used to confirm these assignments. Additionally, cluster ions M+16 and M+32 were characterized following the same strategy. Ions due to the addition of oxygen and alkyl additions were attributed to the presence of methanol in the mobile phase. For the fast chromatographic separation of fullerenes (less than 3.5 min), a sub-2 μm C18 column and isocratic elution (toluene/methanol, 45:55 v/v) was used. Highly selective-selected ion monitoring (H-SIM) mode (mass resolving power, >12,500 fwhm) was proposed monitoring the two most intense isotope ions in the [M](-•) cluster. Method limits of quantitation down to 10 pg L(-1) for C(60) and C(70) fullerenes and between 0.75 and 5.0 ng L(-1) for larger fullerenes were obtained. Finally, the ultrahigh performance liquid chromatography (UHPLC)-APPI-MS method was used to analyze fullerenes in river and pond water samples.

Atmospheric pressure photoionization mass spectrometry of fullerenes.

Analytical strategies for screening, quantitation and confirmation of a group of 100 pesticides in fruit and vegetable samples by LC-MS and LC-MS/MS were developed. The pesticides studied belong to different chemical families of herbicides, insecticides and fungicides. A selection of some degradation products was also included. Chromatographic separation was performed using a Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm and 5 μm particle size), and gradient elution with acetonitrile-water (both with 0.1% formic acid) as mobile phase. LC-MS/MS using highly-selective selected reaction monitoring (H-SRM) acquisition mode monitoring two transitions for each compound showed to be the most sensitive methodology. Quantitation was carried out using matrix-matched standard calibration and good linearity of response was demonstrated (r>0.998). Limits of detection (by acquiring two transitions and with the ion-ratio requirements) ranged between 0.01 and 20 μg/kg were obtained. So, in general, the sensitivity achieved meets the maximum residue levels (MRLs) established by the European Union regulation for food monitoring programs. Pesticide confirmation was carried out following European Union guidelines. In order to prevent false-positives, further confirmatory strategies were proposed. LC-MS in highly-selective selected ion monitoring (H-SIM) mode with accurate mass measurement was used to obtain an orthogonal criterion (exact mass) for confirmation. Accurate mass measurements were always bellow 0.9 mDa for almost all pesticides studied (similar to those described with TOF instruments). A user reversed energy ramp (RER) product ion scan spectra library was generated by means of a data dependent analysis for routine library searching of pesticides. The combination of LC-MS/MS in H-SRM mode and the generation of the RER product ion scan spectra and library search were then used to achieve further confirmation on pesticide analysis. The LC-MS and LC-MS/MS strategies developed were successfully applied for the analysis and confirmation of pesticides in different types of fruit and vegetables samples, and examples of the screening, quantitation and confirmation of pesticides in these samples are shown in this work.

Strategies for the multi-residue analysis of 100 pesticides by liquid chromatography-triple quadrupole mass spectrometry.

There is an increasing need of new bio-analytical methodologies with enough sensitivity, robustness and resolution to cope with the analysis of a large number of analytes in complex matrices in short analysis time. For this purpose, all steps included in any bio-analytical method (sampling, extraction, clean-up, chromatographic analysis and detection) must be taken into account to achieve good and reliable results with cost-effective methodologies. The purpose of this review is to describe the state-of-the-art of the most employed technologies in the period 2009-2012 to achieve fast analysis with liquid chromatography coupled to mass spectrometry (LC-MS) methodologies for bio-analytical applications. Current trends in fast liquid chromatography involve the use of several column technologies and this review will focus on the two most frequently applied: sub-2μm particle size packed columns to achieve ultra high pressure liquid chromatography (UHPLC) separations and porous-shell particle packed columns to attain high efficiency separations with reduced column back-pressures. Additionally, recent automated sample extraction and clean-up methodologies to reduce sample manipulation, variability and total analysis time in bio-analytical applications such as on-line solid phase extraction coupled to HPLC or UHPLC methods, or the use of other approaches such as molecularly imprinted polymers, restricted access materials, and turbulent flow chromatography will also be addressed. The use of mass spectrometry and high or even ultra-high resolution mass spectrometry to reduce sample manipulation and to solve ion suppression or ion enhancement and matrix effects will also be presented. The advantages and drawbacks of all these methodologies for fast and sensitive analysis of biological samples are going to be discussed by means of relevant applications.

State-of-the-art in fast liquid chromatography-mass spectrometry for bio-analytical applications.

A collective view of the degradation of monochlorocatechols in fungi is yet to be attained, though these compounds are recognised as key degradation intermediates of numerous chlorinated aromatic hydrocarbons, including monochlorophenols. In the present contribution we have analysed the degradation pathways of monochlorophenols in Aspergillus nidulans using essentially metabolomics. Degradation intermediates herein identified included those commonly reported (e.g. 3-chloro-cis,cis-muconate) but also compounds never reported before in fungi revealing for 4-chlorocatechol and for 3-chlorocatechol unknown degradation paths yielding 3-chlorodienelactone and catechol, respectively. A different 3-chlorocatechol degradation path led to accumulation of 2-chloromuconates (a potential dead-end), notwithstanding preliminary evidence of chloromuconolactones and protoanemonin simultaneous formation. In addition, some transformation intermediates, of which sulfate conjugates of mono-chlorophenols/chlorocatechols were the most common, were also identified. This study provides critical information for understanding the role of fungi in the degradation of chlorinated aromatic hydrocarbons; furthering their utility in the development of innovative bioremediation strategies.

New branches in the degradation pathway of monochlorocatechols by Aspergillus nidulans: a metabolomics analysis.

This review aims to describe the most significant applications of mass spectrometry-based metabolomics in the field of chemical food safety. A particular discussion of all the different analytical steps involved in the metabolomics workflow (sample preparation, mass spectrometry analytical platform and data processing) will be addressed.

Potential of mass spectrometry metabolomics for chemical food safety.

Biological samples such as tissues, blood, or tumors are often complex and harbor heterogeneous populations of cells. Separating out specific cell types or subpopulations from such complex mixtures to study their metabolic phenotypes is challenging because experimental procedures for separation may disturb the metabolic state of cells. To address this issue, we developed a method for analysis of cell subpopulations using stable isotope tracing and fluorescence-activated cell sorting followed by liquid chromatography-high-resolution mass spectrometry. To ensure a faithful representation of cellular metabolism after cell sorting, we benchmarked sorted extraction against direct extraction. While peak areas differed markedly with lower signal for amino acids but higher signal for nucleotides, mass isotopomer distributions from sorted cells were generally in good agreement with those obtained from direct extractions, indicating that they reflect the true metabolic state of cells prior to sorting. In proof-of-principle studies, our method revealed metabolic phenotypes specific to T cell subtypes, and also metabolic features of cells in the committed phase of the cell division cycle. Our approach enables studies of a wide range of adherent and suspension cell subpopulations, which we anticipate will be of broad importance in cell biology and biomedicine.

Article	Year
Told through the wine: A liquid chromatography-mass spectrometry interplatform comparison reveals the influence of the global approach on the final annotated metabolites in non-targeted meta... Journal of chromatography. A\| PubMed ID: 26795279	2016
Liquid chromatography/multi-stage mass spectrometry of bisphenol A and its halogenated derivatives. Rapid communications in mass spectrometry : RCM\| PubMed ID: 18022958	2007
Field-amplified sample injection-micellar electrokinetic capillary chromatography for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers and derivatives in canned soft dri... Electrophoresis\| PubMed ID: 20422633	2010
Analysis of UV ink photoinitiators in packaged food by fast liquid chromatography at sub-ambient temperature coupled to tandem mass spectrometry. Journal of chromatography. A\| PubMed ID: 21183186	2011
Degradation pathway of pentachlorophenol by Mucor plumbeus involves phase II conjugation and oxidation-reduction reactions. Journal of hazardous materials\| PubMed ID: 22074894	2011
New trends in fast liquid chromatography for food and environmental analysis. Journal of chromatography. A\| PubMed ID: 22153282	2012
Atmospheric pressure photoionization mass spectrometry of fullerenes. Analytical chemistry\| PubMed ID: 22594299	2012
Strategies for the multi-residue analysis of 100 pesticides by liquid chromatography-triple quadrupole mass spectrometry. Journal of chromatography. A\| PubMed ID: 22748376	2012
State-of-the-art in fast liquid chromatography-mass spectrometry for bio-analytical applications. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences\| PubMed ID: 23375883	2013
New branches in the degradation pathway of monochlorocatechols by Aspergillus nidulans: a metabolomics analysis. Journal of hazardous materials\| PubMed ID: 24509097	2014
Potential of mass spectrometry metabolomics for chemical food safety. Bioanalysis\| PubMed ID: 25558941	2015
Metabolite Profiling and Stable Isotope Tracing in Sorted Subpopulations of Mammalian Cells. Analytical chemistry\| PubMed ID: 26855138	2016

Hector Gallart-Ayala

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