Tony Teav (Presenter)
University of Lausanne
Bio: Research technician at the Metabolomics Platform, affiliated to the University of Lausanne. My expertise is in biochemistry and cellular and molecular biology with a focus on the mass spectrometry-based method development for metabolome analyses of complex biological matrices. I hold a BSc in Biochemistry and MSc in Cell Engineering from the University of Lorraine, and I've gained the expertise in liquid chromatography coupled to mass spectrometry during my Master’s training at the Nestlé Institute of Health Sciences.
Authorship: Rayane Mohamed (1), Tony Teav (1), Florence Mehl (1), Hugues Henry (2) & Julijana Ivanisevic (1)
(1) University of Lausanne, Lausanne, Switzerland (2) Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
| Short Abstract Targeted approach was successfully applied to metabolomics although mainly focused on specific classes of compounds. A more diverse and global coverage is sought to determine the affected pathways and better understand metabolic disease mechanisms. In this study a comprehensive HILIC tandem MS platform has been designed to target metabolites from multiple central carbon pathways. A set of 619 metabolites standards was used to build up the library of transition states for MRM. Chromatographic conditions, including stationary phases, mobile phases composition and pH, were evaluated for optimal separation. As a proof of principle, different biological matrices were characterized using this approach. |
Long Abstract
Introduction
Targeted detection is one of the most commonly and successfully used detection modes in mass spectrometry-based metabolomics [1]. It has already made a significant impact in biomarker discovery and cancer metabolism research [2-3] due to its high reproducibility, sensitivity and unambiguous identification. However, this approach is still mainly used to quantify specific classes of compounds that are known as biomarkers of metabolic diseases like amino acids, acylcarnitines, steroids and bile acids. Several attempts were performed to expand the metabolite coverage to major biochemical pathways mainly by using global search of precursors and generation of MRM transitions [4]. A simultaneous screening of essential biochemical pathways may help to go beyond biomarkers and elucidate the mechanisms underlying different metabolic perturbations and diseases. Here we present a high-throughput metabolomics platform for targeted quantification of several hundred metabolites from central carbon pathways. A standard library containing 619 metabolites of biological interest was used to optimize the chromatographic and mass spectrometry parameters. The LC/MS/MS approach was based on a hydrophilic interaction separation and positive/negative ESI detection mode. Multiple Reaction Monitoring (MRM) transitions were optimized for each metabolite standard and each set of LC/MS/MS conditions were evaluated via an in-house designed R script. As a proof of principle, this approach was applied to comprehensively screen the metabolome of diverse biofluids, cell and tissue extracts.
Method
A mass spectrometry metabolite library (MSMLS, Sigma Aldrich) containing 619 unique small molecules was used to prepare 56 standard mixtures (at final concentration of 62.5 ng/mL) for LC/MS/MS method optimization. Complex biological matrices were extracted using 80% MeOH:ACN (1:1, v/v) solvent mixture as a best compromise to efficiently precipitate proteins and extract the maximum of water soluble and lipid metabolites. The extraction was facilitated by vortex (30 sec) and protein precipitation was favored by sample incubation for 1 h at -20°C followed by 15 min centrifugation at 13,000 rpm and 4°C. The resulting supernatant was removed and evaporated to dryness in a vacuum concentrator. The dry extracts were then reconstituted in 100 µL of ACN:H2O (1:1, v/v), sonicated for 10 min, and centrifuged 15 min at 13,000 rpm and 4°C. Finally, the supernatants were transferred to HPLC vials and stored at -80°C prior to LC/MS analysis. HILIC UPLC columns (Waters, Acquity BEH HILIC and BEH Amide) were tested for chromatographic separation. Electrospray ionization MS was performed in positive and negative polarity in MRM detection mode. LC/MS/MS analyses were performed using the 1290 LC and 6495 Triple Quadrupole system (Agilent Technologies, USA). Data were pre-processed using MassHunter Qualitative Analysis software (version B.07.00) and further analyzed with an in-house designed R script to efficiently extract intensities and retention times of each compound of interest and score different LC/MS conditions.
Results
Here we propose a comprehensive targeted metabolomics platform for the accurate measurement of several hundred metabolites from central carbon pathways. Our metabolite library represents one of the largest analyte sets available and consists of 619 authentic metabolite standards, including amino acids, other carboxylic acids, purine and pyrimidine metabolites, sugars, energy currency metabolites (including redox-electron carriers) and other essential, primary metabolites, intermediates in core metabolic pathways. This implied a careful choice of the chromatographic separation to cover the broadest range of analytes taking into account their physico-chemical properties. The choice of HILIC retention mechanism columns was based on the polarity and pKa of the targeted analytes. Indeed, most of the metabolites present in our library were rather polar having a LogP values below zero. Detection of some vitamins might suffer in these conditions but HILIC was chosen as the best global compromise. Several stationary phases were evaluated along with different mobile phases, playing on their composition, pH and nature. Transition states were optimized using an automatic MRM transition optimizer (Agilent, version B.08.00) allowing for fast and efficient screening of the entire metabolite library. The metabolite coverage was further enhanced by combining the ESI detection in positive and negative mode. An in-house designed R script was used to rapidly and efficiently extract relevant information (i.e., intensities, retention time) for metabolite quantification and assess the reproducibility of the LC/MS/MS conditions. Following the selection of conditions providing the best coverage a dynamic MRM method was set up to allow for fast acquisition of high-quality data. This standard-optimized approach was then used to unambiguously investigate the metabolome coverage across different types of biological samples, comprising biofluids (plasma, urine and CSF), cell and tissue extracts. As a warrant for high quality data the analytical sequence contained a standard mixture and a pooled biological QC samples, to control for potential instrumental drift. Each biological matrix was extensively characterized and compared using the relative abundance of different classes of metabolites present.
Conclusion
A high-throughput, multiple pathway targeted metabolomics workflow was developed to measure several hundred unique central carbon metabolites with high selectivity and sensitivity using the hydrophilic interaction chromatography and dynamic MRM analysis. To our knowledge this approach represents one of the first attempts to extensively characterize metabolic changes in different types of biofluids, cell and tissue extracts while simultaneously measuring highly diverse intermediates involved in core biochemical pathways.
References & Acknowledgements:
[1] Yuan, M.; Breitkopf, S. B.; Yang, X.; Asara, J. M. Nat Protoc. 2012,7, 872-81.
[2] Carroll, P. A.; Diolaiti, D.; McFerrin, L.; Gu, H.; Djukovic, D.; Du, J.; Cheng, P. F.; Anderson, S.; Ulrich, M.; Hurley, J.B.; Raftery, D.; Ayer, D. E.; Eisenman, R. N. Cancer Cell 2015, 27, 271-285.
[3] Griffin, J. L. Curr. Opin. Chem. Biol. 2003, 7, 648-654.
[4] Gu, H.; Zhang, P.; Zhu, J.; Raftery, D. Anal. Chem. 2015, 87, 12355-62.
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |