Development of a method for enhancing metabolomics coverage of human sweat by gas chromatography–mass spectrometry in high resolution mode

Sweat has recently gained popularity as clinical sample in metabolomics analysis as it is a non-invasive biofluid the composition of which could be modified by certain pathologies, as is the case with cystic fibrosis that increases chloride levels in sweat. However, the whole composition of sweat is still unknown and there is a lack of analytical strategies for sweat analysis. The aim of the present study was to develop and validate a method for metabolomic analysis of human sweat by gas chromatography–time of flight/mass spectrometry (GC–TOF/MS) in high resolution mode. Thus, different sample preparation strategies were compared to check their effect on the profile of sweat metabolites. Sixty-six compounds were tentatively identified by the obtained MS information. Amino acids, dicarboxylic acids and other interesting metabolites such as myo-inositol and urocanic acid were identified. Among the tested protocols, methyoxiamination plus silylation after deproteinization was the most suited option to obtain a representative snapshot of sweat metabolome. The intra-day repeatability of the method ranged from 0.60 to 16.99% and the inter-day repeatability from 2.75 to 31.25%. As most of the identified metabolites are involved in key biochemical pathways, this study opens new possibilities to the use of sweat as a source of metabolite biomarkers of specific disorders.     

CE-MS for metabolomics: Developments and applications in the period 2018–2020

Capillary electrophoresis-mass spectrometry (CE-MS) is now a mature analytical technique in metabolomics, notably for the efficient profiling of polar and charged metabolites. Over the past few years, (further) progress has been made in the design of improved interfacing techniques for coupling CE to MS; also, in the development of CE-MS approaches for profiling metabolites in volume-restricted samples, and in strategies that further enhance the metabolic coverage. In this article, which is a follow-up of a previous review article covering the years 2016–2018 (Electrophoresis 2019, 40, 165–179), the main (technological) developments in CE-MS methods and strategies for metabolomics are discussed covering the literature from July 2018 to June 2020. Representative examples highlight the utility of CE-MS in the fields of biomedical, clinical, microbial, plant and food metabolomics. A complete overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, some general conclusions and perspectives are given.     

Metabolite mass spectrometry profiling of cacao genotypes reveals contrasting resistances to Ceratocystis cacaofunesta phytopathogen

Ceratocystis wilt is a lethal disease of cacao, and the search for resistant genotypes may provide the best way to deal with the disease. Resistance or susceptibility behavior of some cacao genotypes when infected by Ceratocystis cacaofunesta is not yet understood. Herein, we report an LC-MS metabolomic screening analysis based on high-resolution MS to obtain comprehensive metabolic profile associated with multivariate data analysis of PLS-DA, which was effective to classify CCN-51 and TSH-1188 as resistant genotypes to C. cacaofunesta fungus, while CEPEC2002 was classified as a susceptible one. Using reversed-phase LC method, electrospray interface, and high-resolution tandem MS by the quadrupole-TOF analyzer, the typical profiles of metabolites, such as phenylpropanoids, flavonoids, lipids, alkaloids, and amino acids, were obtained. Untargeted metabolite profiles were used to construct discriminant analysis by partial least squares (PLS-DA)-derived loading plots, which placed the cacao genotypes into two major clusters related to susceptible or resistant groups. Linolenic, linoleic, oleic, stearic, arachidonic, and asiatic acids were annotated metabolites of infected, susceptible, and resistant genotypes, while methyl jasmonate, jasmonic acid, hydroxylated jasmonic acid, caffeine, and theobromine were annotated as constituents of the resistant genotypes. Trends of these typical metabolites levels revealed that CCN51 is susceptible, CEPEC2002 is moderately susceptible, and TSH1188 is resistant to C. cacaofunesta. Therefore, profiles of major metabolites as screened by LC-MS offer an efficient tool to reveal the level of resistance of cacao genotypes to C. cacaofunesta present in any farm around the world.     

木香烃内酯诱导乳腺癌MCF-7细胞凋亡作用机制的研究

研究了木香烃内酯诱导人乳腺癌细胞MCF-7细胞凋亡的作用机制.采用流式细胞仪测定不同浓度木香烃内酯(0,2,4,8 μg/mL)作用于MCF-7细胞后细胞凋亡、活性氧(Reactive oxygen species,ROS)含量及线粒体跨膜电位(Mitochondrial transmembrane potential,MTP)的变化,气相色谱-质谱联用(GC-TOF/MS)技术分析加药组与未加药组的代谢差异物.结果表明,木香烃内酯能诱导MCF-7细胞凋亡,并具有浓度依赖性,能够促使ROS含量升高;MTP在2μg/mL木香烃内酯作用时升高,在4和8μg/mL时显著下降;基于GC-TOF/MS的细胞代谢组学研究,最终发现15种代谢差异物.基于上述结果,推测木香烃内酯通过引起ROS含量升高、MTP降低,扰乱线粒体的正常功能,进一步阻碍TCA循环,抑制ATP合成,扰乱了细胞内代谢物的平衡,并引起位于膜间隙的凋亡相关蛋白释放,最终导致MCF-7细胞的凋亡.     

Development of high throughput 96-blade solid phase microextraction-liquid chromatrography-mass spectrometry protocol for metabolomics

In metabolomics, the workflow for quantitative and comprehensive metabolic mapping of cellular metabolites can be a very challenging undertaking. Sampling and sample preparation play a significant role in untargeted analysis, as they may affect the composition of the analyzed metabolome. In the current work, different solid phase microextraction (SPME) coating chemistries were developed and applied to provide simultaneous extraction of a wide range of both hydrophobic and hydrophilic cellular metabolites produced by a model organism, Escherichia coli. Three different LC-MS methods were also evaluated for analysis of extracted metabolites. Finally, over 200 cellular metabolites were separated and detected with widely varying hydrophobicities ranging within −7 < log P < 15, including amino acids, peptides, nucleotides, carbohydrates, polycarboxylic acids, vitamins, phosphorylated compounds, and lipids such as hydrophobic phospholipids, prenol lipids, and fatty acids at the stationary phase of the E. coli life cycle using the developed 96-blade SPME-LC-MS method.     

HILIC-MS/MS method development for targeted quantitation of metabolites: practical considerations from a clinical diagnostic perspective

The development of targeted assays for polar molecules is a persistent challenge in quantitative metabolite measurement. In addressing these challenges, hydrophilic interaction liquid chromatography (HILIC) has proved to be a valuable, though under-used and poorly understood chromatographic technique. This work has addressed a number of components that are intrinsic in development of a high-throughput, specific and sensitive assay for metabolites using HILIC-MS/MS. Generally accepted HILIC doctrine, such as addition of water to all mobile phases and re-equilibration time, was shown to be flawed under gradient HILIC mode. The effect of non-classical mobile phase additives on HILIC-MS/MS specificity, sensitivity and assay throughput was shown for endogenous metabolites. A broad evaluation of columns and mobile phases for the retention of varied molecular classes was performed, elucidating the empirical nature of HILIC method development. Application of the empirical evaluations performed in the paper was demonstrated by detailing a method development cycle for methylmalonic acid to achieve a highly selective and sensitive HILIC-MS/MS quantitation capable of high-throughput analysis for clinical utility.     

Informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography and high-resolution mass spectrometry (GCxGC-HRMS)

This paper describes informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography (GCxGC) and high-resolution mass spectrometry (HRMS). GCxGC-HRMS analysis produces large data sets that are rich with information, but highly complex. The size of the data and volume of information requires automated processing for comprehensive cross-sample analysis, but the complexity poses a challenge for developing robust methods. The approach developed here analyzes GCxGC-HRMS data from multiple samples to extract a feature template that comprehensively captures the pattern of peaks detected in the retention-times plane. Then, for each sample chromatogram, the template is geometrically transformed to align with the detected peak pattern and generate a set of feature measurements for cross-sample analyses such as sample classification and biomarker discovery. The approach avoids the intractable problem of comprehensive peak matching by using a few reliable peaks for alignment and peak-based retention-plane windows to define comprehensive features that can be reliably matched for cross-sample analysis. The informatics are demonstrated with a set of 18 samples from breast-cancer tumors, each from different individuals, six each for Grades 1-3. The features allow classification that matches grading by a cancer pathologist with 78% success in leave-one-out cross-validation experiments. The HRMS signatures of the features of interest can be examined for determining elemental compositions and identifying compounds.     

Urinary signature of anabolic steroids and glucocorticoids in humans by LC-MS

A metabonomic strategy based on LC-MS was employed to investigate the metabolic profile of urine samples from 20 athletes who had been tested positive for corticoids and anabolic steroids and 29 controls. In this aim, different sample preparations and chromatographic conditions were compared. The acquired LC-MS data of doped athletes and controls were subjected to analysis of variance (ANOVA) and principal component analysis (PCA). Using this approach, molecular signature of human urine was obtained showing that metabonomics could be a complementary tool to discriminate different urinary profiles and to track down metabolic changes in humans.     

Solid-phase microextraction. How far are we from clinical practice?

The current review briefly discusses the future development of solid-phase microextraction (SPME) and its potential application in the field of clinical medicine, including pharmacokinetic studies, therapeutic drug monitoring, biomarker discovery, and targeted and untargeted metabolomics. We also discuss aspects of automation and high-throughput analysis as major requirements of daily clinical practice. We give examples of clinically-validated applications of SPME and point out the regulatory restrictions limiting some in-vivo SPME studies. We briefly review the current state of progress in this extraction technique in the context of its future application in medical research and laboratory testing, including new directions (i.e. personalized medicine).     

Effects of ultrasound-assisted thawing on lamb meat quality and oxidative stability during refrigerated storage using non-targeted metabolomics

The aim of this study was to evaluate the changes of ultrasound-assisted thawing on lamb meat quality and differential metabolite profiles during refrigerated storage. Compared with flow water thawing (FW), pH, a*, C*, and sulfhydryl content of lamb were significantly increased, while L*, drip loss and cooking loss were significantly decreased after ultrasound-assisted thawing (UT). On day 1 (UT1 and FW1) and day 7 (UT7 and FW7) in the UT and FW groups, principal component analysis explained 42.22% and 39.25% of the total variance. In this study, 44 (UT1 and FW1) and 47 (UT7 and FW7) differentially expressed metabolites were identified, including amino acids, carbohydrates and their conjugates, nucleic acids, carbonyl compounds and others. The results of this study provide data to clarify the differences between UT and FW, and lay a foundation for the application of ultrasound-assisted thawing in the meat industry.