bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2019‒11‒03
twenty-four papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Sep Sci. 2019 Nov 01.
      A lyophilization-supercritical fluid extraction coupled with supercritical fluid chromatography-MS/MS online method was developed for the determination of lipid mediators in breast cancer cells. Supercritical fluid extraction was applied to the cell samples for the first time due to the use of lyophilization. The conditions of supercritical fluid extraction and supercritical fluid chromatography-MS/MS were investigated systematically. Under the optimized conditions, all the calibration curves for the lipid mediators showed good linearity (R2  > 0.99). The limits of detection and the limits of quantification were in the range of 0.190-5.36 pg and 0.560-16.2 pg, respectively. The recoveries were in the range of 70.3%-125%. The relative standard deviations of the precision ranged from 1.49%-18.7% and the accuracies were higher than 84%. Compared with liquid-liquid extraction coupled with LC-MS/MS method, the present approach reduced the manual labor and obtained higher sensitivity as well as higher extraction recoveries for all 15 lipid mediators. Finally, the online method was applied to the quantification of lipid mediators in breast cancer cells and normal mammary epithelial cells. On the basis of the results, this lyophilization-supercritical fluid extraction online coupled with supercritical fluid chromatography-MS/MS method showed great promise in the analysis of lipid mediators in complex biological samples. This article is protected by copyright. All rights reserved.
    Keywords:  breast cancer cells; lipid mediators; lyophilization; supercritical fluid chromatography; supercritical fluid extraction
  2. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Oct 04. pii: S1570-0232(19)30754-8. [Epub ahead of print]1129 121805
      Catecholamines [dopamine (DA), epinephrine (E), and norepinephrine (NE)] and their metabolites [metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT)] are functionally important in humans. Their overexpression can indicate the presence of neuroendocrine tumors. Accurate and rapid quantitation of catecholamines and their metabolites may function in differential diagnosis of neuroendocrine tumors. Herein, we diluted 200 μL plasma using isotope labelled internal standards (IS), and extracted using solid phase extraction. The performance of isotope diluted liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) was evaluated and applied to quantify the level of catecholamines and metabolites in clinical samples from 73 apparently healthy adults. The total analysis time of the ID-LC-MS/MS method was 4 min. The improved method was highly sensitive, with a limit of quantification (LOQ) for MN, NMN, 3-MT, and E of 1 pg/mL, a LOQ for DA of 5 pg/mL, and for NE of 10 pg/mL. After correction using IS, no significant matrix effects were observed. Good reproducibility was obtained, with total CVs of 3.2-13.1% (DA), 4.8-10.0% (E), 6.2-6.9% (NE), 3.8-7.9% (MN), 4.1-8.8% (NMN), 3.4-8.9% (3-MT). Recoveries were in the range of 91.1-109.7% for the six analytes. Also, the mean concentration of catecholamines were as follows: MN, 22.9 ± 7.2 pg/mL; NMN, 41.4 ± 17.2 pg/mL; 3-MT, 2.34 ± 2.01 pg/mL; DA, 10.2 ± 4.6 pg/mL; E, 29.3 ± 14.2 pg/mL and NE 427.0 ± 190.6 pg/mL. A reliable ID-LC-MS/MS method for the determination of catecholamines and their metabolites using small volumes of plasma was verified. This method is rapid, simple, and may serve as an essential diagnostic tool for neuroendocrine tumors in clinical practice.
    Keywords:  Catecholamine; Liquid chromatography-tandem mass spectrometry; Metanephrine
  3. Rapid Commun Mass Spectrom. 2019 Nov 02.
      RATIONALE: The activity of the glucocorticoid activating enzyme, 11β-hydroxysteroid dehydrogenase type-1 (11βHSD1) is altered in diseases such as obesity, inflammation and psychiatric disorders. In rodents 11βHSD1 converts inert 11-dehydrocorticosterone (11-DHC), to the active form, corticosterone (CORT). A sensitive, specific liquid chromatography-tandem mass spectrometry method was sought to simultaneously quantify total 11-DHC and total and free CORT in murine plasma for simple assessment of 11βHSD1 activity in murine models.METHODS: Mass Spectrometry parameters were optimised and the chromatographic separation of CORT and 11-DHC was developed. Murine plasma was prepared by 10:1 chloroform liquid-liquid extraction (LLE) for analysis. Limits of Quantitation (LOQs), linearity and other method criteria were assessed, according to bioanalytical method validation guidelines.
    RESULTS: Reliable separation of 11-DHC and CORT was achieved, using an ACE Excel 2 C18-AR (2.1 x 150 mm; 2 󠇆μm) fused core column at 25o C, with an acidified water; acetonitrile gradient over 10 minutes. Analytes were detected by multiple reaction monitoring after positive electrospray ionization (m/z 345.1.1➔ 121.2, m/z 347.1➔121.1 for 11-DHC and CORT, respectively). The LOQs were 0.25 and 0.20 ng/mL for 11-DHC and CORT, respectively.
    CONCLUSION: This LC/MS method is suitable for the reliable analysis of 11-DHC and CORT following simple LLE of murine plasma, bringing preclinical analysis in line with recommendations for clinical endocrinology and biochemistry.
  4. Biomed Chromatogr. 2019 Oct 27. e4723
      Abnormal production or metabolism of steroid hormones are responsible for the development of endocrine diseases. The accurate quantification of steroid hormones is needed for both research into clinical conditions as well as for diagnostic and monitoring purposes. The improved analytical method for 39 steroid profiling in urine was developed using LC-MS/MS. As a pre-treatment procedure prior to LC-MS/MS analysis, hydrolysis using β-glucuronidase and solid-phase extraction for purifying the samples were performed. Steroids were separated using Waters ACQUITY @ BEH C18 column (2.1 x 100 mm, 1.7 μm) and a mobile phase consisting of eluent A (0.01% formic acid and 1 mM ammonium formate in water) and eluent B (0.01% formic acid and 1 mM ammonium formate in methanol) with a gradient program at a flow rate of 0.4 mL/min. Under the optimized method, the linearity of calibration curves was higher than 0.992. The limits of detection (LOD) at signal to noise (S/N) ratio of 3 were 0.03-90 ng/mL. The developed novel LC-MS/MS method can quantitatively profile 39 steroids in a single analytical run. Steroid profiling based on quantitative results could improve the diagnosis and monitoring of hormone-dependent diseases.
    Keywords:  LC-MS/MS; Metabolomics; Steroid profiling; quantitatively profile
  5. Rapid Commun Mass Spectrom. 2019 Oct 31.
      RATIONALE: Methylated amino compounds and basic amino acids are important analyte classes with high relevance in nutrition, physical activity and physiology. Reliable and easy quantification methods covering a variety of metabolites in body fluids are a prerequisite for efficient investigations in the field of food and nutrition.METHODS: Targeted UHPLC/MS analysis was performed using HILIC separation and timed ESI-MRM detection, combined with a short sample preparation. Calibration in urine and blood plasma was achieved by matrix-matched standards, isotope-labelled internal standards and standard addition. The method was fully validated and the performance was evaluated using a subset from the Karlsruhe Metabolomics and Nutrition (KarMeN) study.
    RESULTS: Within this method, a number of 30 compounds could be quantified simultaneously in a short run of 9 minutes in both body fluids. It covers a variety of free amino compounds which are present in very different concentrations. The method is easy, precise and robust, and has a broad working range. As a proof of principle, literature-based associations of certain metabolites with dietary intake of respective foods were clearly confirmed in the KarMeN subset.
    CONCLUSIONS: Overall, the method turned out to be well suited for application in nutrition studies, as shown for the example of food intake biomarkers in KarMeN. Application to a variety of questions such as food-related effects, or physical activity will support future studies in the context of nutrition and health.
  6. Rapid Commun Mass Spectrom. 2019 Nov 02.
      Rationale Melamine is ubiquitously present in our daily life. It has a known effect on the kidneys, but it may also adversely affect the reproduction system. We developed an analytical method of measuring melamine levels in maternal placenta and correlated these levels with melamine concentrations in urine, a necessary step in finding out if melamine might cross the placenta and enter the circulation of the fetus.METHODS: We used liquid-liquid extraction, clean up by solid phase extraction (SPE), and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) to measure melamine in placenta specimens. The results of this method were assessed for linearity, limits of quantitation (LOQ), and intra- and inter-assay precision as well as accuracy, matrix effect, and recovery rate.
    RESULTS: Calibration curves indicated good linearity (r>0.995) over concentrations ranging from 5 to 500 ng/mL in placenta specimens, intra- and inter-assay precision from 0.89% to 27.07%, and accuracy from 92.4% to123.5%. Recovery ranged from 63.9 to 83.9%, and the LOQ was 5 ng/mL in placenta (0.2 g). Placental melamine levels ranged from 7.87 to19.64 ng/mL, all detectable (n = 8). Pregnant women with higher levels of urinary melamine had higher placenta melamine levels than those with non-detectable urinary melamine, though, the results were not significantly different (p=0.149, n=4 in each group).
    CONCLUSIONS: The results of this study suggest that pregnant women were exposed low doses of melamine in daily life as measured urine samples and placenta specimens. It is unclear whether placenta melamine concentrations can better represent long-term exposure than urine or whether melamine in uterus can enter the fetus via this route.
  7. J Anal Methods Chem. 2019 ;2019 2980596
      Ultrahigh-performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (Q/TOF-MS) in the MS/MS mode and UPLC coupled with triple quadrupole mass spectrometry (QqQ-MS) using the multiple reaction monitoring (MRM) mode were used to make a qualitative and quantitative analysis of twelve bile acids in Bile Arisaema. The fragmentation pathway of twelve bile acids was proposed. The quantification method showed a good linearity over a wide concentration range (R 2 > 0.99), repeatability (RSD < 4.12%), stability (RSD < 4.25%), precision (RSD < 4.06%), and recovery (95.36-102.15%). Content of twelve compounds in Bile Arisaema varied significantly depending on region. Chemometric methods, hierarchical clustering analysis (HCA), and principal components analysis (PCA) were successfully used to optimize the fermentation time of the Bile Arisaema. The results suggested that the Bile Arisaema could complete fermentation in 15 days. The possible processing mechanism of Bile Arisaema promoted the transformation of conjugated bile acids into free bile acids in fermentation.
  8. Nutr Hosp. 2019 Oct 28.
      Vitamin D is a micronutrient that plays a large role in bone disease, and researchers are now discovering that it also does so in non-skeletal disease, thus making high-quality analytical determination necessary. To make this determination, a series of immunochemical and physical methods are used. These methods present a series of different ways of handling samples as well as different methodologies that bring a series of advantages and limitations based on the scope of work in which the vitamin D analysis methodology is applied. Although the Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) is the gold standard method of analytical vitamin D determination, and is the only one to offer a more complete and accurate view of all metabolites of this vitamin, it is necessary to standardize all the analysis methodologies that allow accurate, reliable and quality analytical determination, since it is essential to obtain results that can reliably be extrapolated to the population, and that can be decisive in assessing a large number of pathologies.
  9. Steroids. 2019 Oct 28. pii: S0039-128X(19)30221-1. [Epub ahead of print] 108531
      Steroids are an important biomolecule class for analysis due to their promise as biomarkers for various diseases and their abuse as performance enhancers in sports. Current analytical methods, including chromatography and nuclear magnetic resonance spectroscopy, fall short of being able to confidently analyze steroids, partly due to the large number of steroid isomers. Ion mobility spectrometry (IMS), a gas-phase ion separator, has shown potential for steroid analysis both in conjunction with liquid chromatography (LC) and as a stand-alone technique. This review will examine the current literature on IMS analysis of steroids. Analysis by LC-IMS will include examination of steroids and steroid glucuronides in human urine and serum samples for enhanced signal-to-noise ratios and higher confidence of identification. The stand-alone IMS analysis will examine the use of derivatization of steroids and formation of multimers to enhance resolution for steroid isomers analysis, where both methods have shown to greatly increase the separation of steroid isomer species. However, these methods have not been applied to biological mixtures to assess their applicability to medical and forensic applications, which should be a future direction of this field.
    Keywords:  Ion Mobility Spectrometry; Liquid Chromatography; Steroid Analysis
  10. J Clin Endocrinol Metab. 2019 Oct 29. pii: dgz141. [Epub ahead of print]
      CONTEXT: Urine steroid metabolomics, combining mass spectrometry-based steroid profiling and machine learning, has been described as a novel diagnostic tool for detection of adrenocortical carcinoma (ACC).OBJECTIVE, DESIGN, SETTING: This proof-of-concept study evaluated the performance of urine steroid metabolomics as a tool for post-operative recurrence detection after microscopically complete (R0) resection of ACC.
    PATIENTS AND METHODS: 135 patients from 14 clinical centers provided post-operative urine samples, which were analyzed by gas chromatography-mass spectrometry. We assessed the utility of these urine steroid profiles in detecting ACC recurrence, either when interpreted by expert clinicians, or when analyzed by Random Forest, a machine learning-based classifier. Radiological recurrence detection served as the reference standard.
    RESULTS: Imaging detected recurrent disease in 42 of 135 patients; 32 had provided pre- and post-recurrence urine samples. 39 patients remained disease-free for ≥3 years. The urine "steroid fingerprint" at recurrence resembled that observed before R0 resection in the majority of cases. Review of longitudinally collected urine steroid profiles by three blinded experts detected recurrence by the time of radiological diagnosis in 50-72% of cases, improving to 69-92%, if a pre-operative urine steroid result was available. Recurrence detection by steroid profiling preceded detection by imaging by more than 2 months in 22-39% of patients. Specificities varied considerably, ranging from 61 to 97%. The computational classifier detected ACC recurrence with superior accuracy (sensitivity=specificity=81%).
    CONCLUSION: Urine steroid metabolomics is a promising tool for post-operative recurrence detection in ACC; availability of a pre-operative urine considerably improves the ability to detect ACC recurrence.
    Keywords:  ACC; adrenocortical carcinoma; machine learning; mass spectrometry; recurrence detection; steroid metabolomics
  11. Rapid Commun Mass Spectrom. 2019 Oct 31.
      RATIONALE: Hepcidin is a peptide hormone that plays a central role in regulating iron metabolism. It is a potential biomarker for the diagnosis, monitoring and treatment of iron metabolism disorders. Serum hepcidin level can differ by 3 orders of magnitude depending on the patient's condition. Existing LC/MS assays lack clinical sensitivity or require costly sample preparation steps. A simple, sensitive, robust, and cost-effective assay for serum hepcidin quantitation in routine clinical laboratories is needed.METHODS: An HPLC/MS/MS method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable isotope labeled hepcidin as internal standard. The method was validated according to CLSI-C62A guidelines. Calibrators were prepared with hepcidin-free serum. Clinical samples were separately processed and compared using solid phase extraction (SPE) and acetonitrile (ACN) protein precipitation.
    RESULTS: The calibration curve was validated over the range of 0.1-100 nmol/L with R2 >0.99. Both SPE and ACN had excellent and comparable reproducibility. The intra-day and inter-day CVs were <3% and <6%,. There was 89% and 88% hepcidin recovery by SPE and ACN preparation. Measurement of secondary reference material using non-traceable calibrators yielded up to 30% positive bias, comparable with values obtained by an external comparator. Hepcidin was stable in serum at ambient temperature and at 4°C. Relative errors (RE) were ≤1.2% and ≤4.4%, respectively. Freeze-thaw (-70°C) stability after 3 cycles showed a RE of ≤1.8%. The impact on hepcidin recovery due to hemolysis (4+), lipemia (4+) and Icterus (4+) was < 3%.
    CONCLUSION: We have developed and validated a simple, sensitive, robust, and cost-effective HPLC/MS/MS method for the quantitation of serum hepcidin. The method uses ACN protein precipitation for sample preparation and reverse-phase normal flow HPLC. Sample preparation is inexpensive; it can be automated with a liquid handling system to allow high-throughput application.
  12. Chirality. 2019 Oct 30.
      A novel chiral derivatization reagent, the N-[1-oxo-5-(triphenylphosphonium)pentyl]- (R)-1,3-thiazolidinyl-4-N-hydroxysuccinimide ester bromide salt (OTPTHE), was developed for the separation and selective detection of chiral DL-amino acids by RP-HPLC analysis. The OTPTHE reacted with DL-amino acids at 60°C maintained for 30 minutes in the presence of 100 mM borate buffer (pH 9.5). The separability of the diastereomeric derivatives was evaluated in terms of the resolution value (Rs) using 13 kinds of DL-amino acids, which were completely separated by reversed-phase chromatography using C18 column at 254 nm. The Rs of the DL-amino acids varied from 1.62 to 2.51. As for the application of the DL-amino acids, the determination of DL-Ser in the human plasma of healthy volunteers was performed based on our developed method. It was shown that linear calibrations were available with high coefficients of correlation (r2 > 0.9997). The limit of detection (S/N = 3) of the DL-Ser enantiomers was 5.0 pmol; the relative standard deviations of the intraday and interday variations were below 4.56%; the accuracy ranged between 95.40%-110.06% and 95.45%-109.80%, respectively; the mean recoveries (%) of the DL-Ser spiked in the human plasma were 99.49%-103.74%. The amounts of DL-Ser in the human plasma of healthy volunteers were determined.
    Keywords:  D-serine; DL-amino acid; chiral derivatization reagent; human plasma; thiophosphate structure
  13. J Sep Sci. 2019 Oct 31.
      High-performance liquid chromatography coupled with photodiode array detection has been extensively applied in many fields and the peaks among the analyzed samples can be shifted due to the variations of instrumental and experimental conditions. In the multivariate analysis, the retention time alignment is an important pretreatment step. Hence, the shifted peaks in High-performance liquid chromatography coupled with photodiode array detection three-dimensional spectra should be aligned for further analysis. Being motivated by this purpose, the interval correlated shifting method combined with the proposed data arrangement methods are recommended and employed on High-performance liquid chromatography coupled with photodiode array detection data as a demonstration. We validate the alignment performance of the proposed method through comparison the consistency of the retention time before and after alignment. The obtained results demonstrated that the proposed method is capable of successful aligning the employed data. Additionally, the interval correlated shifting method combined with the data arrangement modes is implemented in an easy-to-use graphical user interface environment and so can be operated easily by users not familiar with programming languages. This article is protected by copyright. All rights reserved.
    Keywords:  Data arrangement; Graphical user interface; Interval correlated shifting; Retention time alignment; Three-dimensional spectra
  14. Adv Clin Chem. 2019 ;pii: S0065-2423(19)30060-5. [Epub ahead of print]93 115-167
      The qualitative and quantitative determination of insulin and its related substances (e. g., C-peptide) is of great importance in many different areas of analytical chemistry. In particular, due to the steadily increasing prevalence of metabolic disorders such as diabetes mellitus, an adequate control of the circulating amount of insulin is desirable. In addition, also in forensics and doping control analysis, the determination of insulin in blood, urine or other biological matrices plays a major role. However, in order to establish general reference values for insulin and C-peptide for diabetology, the comparability of measured concentrations is indispensable. This has not yet been fully implemented, although enormous progress has been made in recent years, and the search for a "gold standard" method is still ongoing. In addition to established ligand-binding assays, an increasing number of mass-spectrometric methods have been developed and employed as the to-date available systems (for example, high-resolution/high accuracy mass spectrometers) provide the sensitivity required to determine analyte concentrations in the sub-ng/mL (sub-100pmol/L) level. Meanwhile, also high-throughput measurements have been realized to meet the requirement of testing a high number of samples in a short period of time. Further developments aim at enabling the online measurement of insulin in the blood with the help of an insulin sensor and, in the following, in addition to a brief review, today's state of the art testing developments are summarized.
    Keywords:  C-peptide; Insulin; Insulin analogues; Mass spectrometry; Standardization
  15. Clin Chem Lab Med. 2019 Oct 30. pii: /j/cclm.ahead-of-print/cclm-2019-0858/cclm-2019-0858.xml. [Epub ahead of print]
      Mass spectrometry (MS) is the workhorse of metabolomics, proteomics and lipidomics. Mass spectrometry imaging (MSI), its extension to spatially resolved analysis of tissues, is a powerful tool for visualizing molecular information within the histological context of tissue. This review summarizes recent developments in MSI and highlights current challenges that remain to achieve molecular imaging at the cellular level of clinical specimens. We focus on matrix-assisted laser desorption/ionization (MALDI)-MSI. We discuss the current status of each of the analysis steps and remaining challenges to reach the desired level of cellular imaging. Currently, analyte delocalization and degradation, matrix crystal size, laser focus restrictions and detector sensitivity are factors that are limiting spatial resolution. New sample preparation devices and laser optic systems are being developed to push the boundaries of these limitations. Furthermore, we review the processing of cellular MSI data and images, and the systematic integration of these data in the light of available algorithms and databases. We discuss roadblocks in the data analysis pipeline and show how technology from other fields can be used to overcome these. Finally, we conclude with curative and community efforts that are needed to enable contextualization of the information obtained.
    Keywords:  data analysis; data integration and interpretation; histopathology; mass spectrometry imaging; molecular analysis; spatial resolution
  16. J Mass Spectrom. 2019 Oct 29.
      While a number of approaches have been developed to analyze liquid chromatography tandem mass spectrometry (LC-MS/MS) data obtained from modified oligonucleotides, the majority of these methods require analyzing every MS/MS spectrum de novo to sequence the oligonucleotide and place the modification. Spectral matching is an alternative approach for analyzing MS/MS data that is based on creating a library of annotated MS/MS spectra against which individual MS/MS data can be searched. Here we have adapted the existing NIST spectral matching software to enable its use in the interpretation of MS/MS data obtained from modified oligonucleotides. In particular, we demonstrate the utility of this approach to identify specific post-transcriptionally modified nucleosides in particular transfer RNAs (tRNAs) obtained through a conventional RNA modification mapping experimental protocol. Spectral matching was found to be an efficient approach for screening for known modified tRNAs by using the experimental data as the library and previously annotated RNase T1 digestion products of tRNAs as the reference spectra. The utility of spectral matching for rapid analysis of multiple LC-MS/MS analyses was demonstrated by screening mutant strains of Streptococcus mutans to identify the enzyme(s) responsible for synthesizing the tRNA position 37 modification threonylcarbamoyladenosine (t6 A).
    Keywords:  LC-MS/MS; RNA sequencing; modified nucleosides; spectral matching; tRNA; tandem mass spectrometry
  17. Methods Enzymol. 2019 ;pii: S0076-6879(19)30257-5. [Epub ahead of print]628 293-307
      Quantitative mass spectrometry analysis of metabolites at a single-cell level is critical to understanding the cell functionality and heterogeneity. To preserve cell viability after extraction, the extracted volume needs to be precisely controlled at a subpicoliter-to-picoliter level. Recently, we developed a volume-controlled, and highly sensitive approach for live cell analysis at a single-cell level by integrating electroosmotic extraction and nano-electrospray ionization mass spectrometry (nanoESI MS) analysis. Herein, we use outer epidermal cells of Allium cepa as a model system to present the details of our workflow, including detailed descriptions of the experimental setup for live cell analysis, preparation of the extraction nanopipette, establishment of calibration curves, and extraction and quantification of glucose in an individual onion cell. The capability of this procedure for quantitative live cell analysis has been demonstrated by accurate quantification of glucose in Allium cepa. In principle, our approach is applicable to identification and quantification of metabolites in live mammalian cells.
    Keywords:  Electroosmotic extraction; Live cell analysis; Metabolite quantification; Nano-electrospray ionization mass spectrometry (nanoESI MS); Single cell
  18. J Mass Spectrom. 2019 Oct 29.
      The mass spectrometry based molecular profiling can be used for better differentiation between normal and cancer tissues as well as for the detection of neoplastic transformation which is of great importance for diagnostics of a pathology, prognosis of its evolution trend and development of a treatment strategy. The aim of the present study is the evaluation of tissue classification approaches based on various datasets derived from the molecular profile of the organic solvent extracts of a tissue. A set of possibilities are considered for the orthogonal partial least squares discriminant analysis: all mass spectrometric peaks over 300 counts threshold; subset of peaks selected by ranking with support vector machine algorithm; peaks selected by random forest algorithm; peaks with the statistically significant difference of the intensity determined by the Mann-Whitney U-test; peaks identified as lipids; both identified and significantly different peaks. The best predictive potential is obtained for OPLS-DA model built on nonpolar glycerolipids (Q2 = 0.64, AUC = 0.95), the second one is OPLS-DA model with lipid peaks selected by random forest algorithm (Q2 = 0.58, AUC = 0.87). Moreover, models based on particular molecular classes are more preferable from biological point of view, resulting in new explanatory mechanisms of pathophysiology and providing a pathway analysis. Another promising features for OPLS-DA modeling are phosphatidylethanolamines (Q2 = 0.48, AUC = 0.86).
    Keywords:  ESI-MS; cervical cancer; discriminant model; feature selection; mass spectrometry; shotgun lipidomics
  19. Anal Chem. 2019 Oct 29.
      Comprehensive and unambiguous identification of small molecules in complex samples will revolutionize our understanding of the role of metabolites in biological systems. Existing and emerging technologies have enabled measurement of chemical properties of molecules in complex mixtures and, in concert, are sensitive enough to resolve even stereoisomers. Despite these experimental advances, small molecule identification is inhibited by (i) chemical reference libraries (e.g., mass spectra, collision cross section, and other measurable property libraries) representing <1% of known molecules, limiting the number of possible identifications, and (ii) the lack of a method to generate candidate matches directly from experimental features (i.e., without a library). To this end, we developed a variational autoencoder (VAE) to learn a continuous numerical, or latent, representation of molecular structure to expand reference libraries for small molecule identification. We extended the VAE to include a chemical property decoder, trained as a multitask network, in order to shape the latent representation such that it assembles according to desired chemical properties. The approach is unique in its application to metabolomics and small molecule identification, with its focus on properties that can be obtained from experimental measurements (m/z, CCS) paired with its training paradigm, which involved a cascade of transfer learning iterations. First, molecular representation is learned from a large dataset of structures with m/z labels. Next, in silico property values are used to continue training, as experimental property data is limited. Finally, the network is further refined by being trained with the experimental data. This allows the network to learn as much as possible at each stage, enabling success with progressively smaller datasets without overfitting. Once trained, the network can be used to predict chemical properties directly from structure, as well as generate candidate structures with desired chemical properties. Our approach is orders of magnitude faster than first-principles simulation for CCS property prediction. Additionally, the ability to generate novel molecules along manifolds, defined by chemical property analogues, positions DarkChem as highly useful in a number of application areas, including metabolomics and small molecule identification, drug discovery and design, chemical forensics, and beyond.
  20. Anal Chem. 2019 Oct 29.
      Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is increasingly used to visualize the chemical communication between microorganisms. However, to fully exploit the potential of this label-free technique, crucial methodological advances are still needed. In particular, with current microbial MALDI-MSI methods chemical coverage is strongly limited to well ionizing compounds and a safe MSI-compatible inactivation of microbial viability and quenching of metabolism is not possible. Here, we introduce a membrane-based culturing workflow that enables a rapid MSI-compatible steam inactivation of pathogens and generation of a flat surface. We equipped precision mass spectrometers with laser-post-ionization (MALDI-2) modules to increase the analytical sensitivity by up to several orders of magnitude. In this way, for example 39 different 2-alkylquinolones with differential expression patterns and a similar number of glycerophospholipids were simultaneously visualized from single cultures of Pseudomonas aeruginosa at about 50 µm resolution. To visualize the metabolic exchange between competing microorganisms, we challenged commensal Escherichia coli MG1655 and virulence factor-depleted E. coli C600 strains with enteropathogenic Shiga-toxin negative E. coli O26:H11, and Staphylococcus aureus with antagonistic P. aeruginosa. Insight into the 3-dimensional organization of a biofilm of the probiotic E. coli Nissle 1917 was obtained after developing an embedding/cryosectioning protocol. Our advanced protocols could help to substantially increase the application range of microbial MS imaging.
  21. J Steroid Biochem Mol Biol. 2019 Oct 28. pii: S0960-0760(19)30638-7. [Epub ahead of print] 105515
  22. Sci Rep. 2019 Oct 30. 9(1): 15690
      Endometriosis is a pathologic condition affecting approximately 10% of women in their reproductive years. Characterized by abnormal growth of uterine endometrial tissue in other body areas, endometriosis can cause severe abdominal pain and/or infertility. Despite devastating consequences to patients' quality of life, the causes of endometriosis are not fully understood and validated diagnostic markers for endometriosis have not been identified. Molecular analyses of ectopic and eutopic endometrial tissues could lead to enhanced understanding of the disease. Here, we apply desorption electrospray ionization (DESI) mass spectrometry (MS) imaging to chemically and spatially characterize the molecular profiles of 231 eutopic and ectopic endometrial tissues from 89 endometriosis patients. DESI-MS imaging allowed clear visualization of endometrial glandular and stromal regions within tissue samples. Statistical models built from DESI-MS imaging data allowed classification of endometriosis lesions with overall accuracies of 89.4%, 98.4%, and 98.8% on training, validation, and test sample sets, respectively. Further, molecular markers that are significantly altered in ectopic endometrial tissues when compared to eutopic tissues were identified, including fatty acids and glycerophosphoserines. Our study showcases the value of MS imaging to investigate the molecular composition of endometriosis lesions and pinpoints metabolic markers that may provide new knowledge on disease pathogenesis.
  23. Int J Mol Sci. 2019 Oct 30. pii: E5416. [Epub ahead of print]20(21):
      Glucagon circulates in concentrations in the low picomolar range, which is demanding regarding the sensitivity of the methods for quantification applied. In addition, the differential and tissue specific proteolytic processing of the glucagon precursor and the presence in of several glucagon-like sequences, not only in the precursor of glucagon, but also in a number of other peptides of the glucagon-secretin family of peptides, put special demands on the specificity of the assays. Finally, experience has shown that unspecific interference of plasma components has presented additional problems. All of these problems have resulted in a lot of diverging results concerning measured and reported glucagon responses in both humans and experimental animals that have and still are causing considerable debate and controversy. There is very solid evidence that glucagon is an important hormone in human and mammalian metabolism, but its precise physiological role in glucose and lipid metabolism and in metabolic disease has been difficult to establish, not least because of these difficulties. It was our purpose with this review to discuss the methods of glucagon quantification and discuss pitfalls and sources of error. We also reviewed some of the dogmas regarding glucagon secretion in the light of the methodological difficulties.
    Keywords:  alpha cell; amino acids; diabetes; glucose; hyperglucagonemia
  24. Methods Enzymol. 2019 ;pii: S0076-6879(19)30252-6. [Epub ahead of print]628 1-17
      Sequential metabolic enzymes have long been hypothesized to form multienzyme metabolic complexes to regulate metabolic flux in cells. Although in vitro biochemistry has not been fruitful to support the hypothesis, advanced biophysical technologies have successfully resurrected the hypothesis with compelling experimental evidence. As biochemistry has always evolved along with technological advancement over the century (e.g., recombinant protein expression, site-directed mutagenesis, advanced spectroscopy and structural biology techniques, etc.), there has been growing interest in advanced imaging-based biophysical methods to explore enzymes inside living cells. In this work, we describe how we visualize two phase-separated biomolecular condensates of multienzyme metabolic complexes that are associated with de novo purine biosynthesis and glucose metabolism in living human cells and how imaging-based data are quantitatively analyzed to advance our knowledge of enzymes and their assemblies in living cells. Therefore, we envision that the framework we describe here would be the starting point to investigate other metabolic enzymes and their assemblies in various cell types with an unprecedented potential to comprehend enzymes and their network in native habitats.
    Keywords:  Fluorescence microscopy; Glucosome; High-content analysis; Live-cell imaging; Metabolic complex; Metabolon; Phase-separated biomolecular condensate; Purinosome; Single cell analysis