extraction-aqueous-metabolites-from-cultured-adherent-cells-for

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Human Metabolome Technologies, Inc

One of the technical challenges encountered during metabolomics research is determining the chemical structures of unidentified peaks. We have developed a metabolomics-based chemoinformatics approach for ranking the candidate structures of unidentified peaks. Our approach uses information about the known metabolites detected in samples containing unidentified peaks and involves three discrete steps. The first step involves identifying "precursor/product metabolites" as potential reactants or products derived from the unidentified peaks. In the second step, candidate structures for the unidentified peak are searched against the PubChem database using a molecular formula. These structures are then ranked by structural similarity against precursor/product metabolites and candidate structures. In the third step, the migration time is predicted to refine the candidate structures. Two simulation studies were conducted to highlight the efficacy of our approach, including the use of 20 proteinogenic amino acids as pseudo-unidentified peaks, and leave-one-out experiments for all of the annotated metabolites with and without filtering against the Human Metabolome Database. We also applied our approach to two unidentified peaks in a urine sample, which were identified as glycocyamidine and N-acetylglycine. These results suggest that our approach could be used to identify unidentified peaks during metabolomics analysis.

Metabolomics-based approach for ranking the candidate structures of unidentified peaks in capillary electrophoresis time-of-flight mass spectrometry.

This study sought to characterize the plasma metabolite profiling of patients with major depressive disorder (MDD).

Plasma metabolome analysis of patients with major depressive disorder.

Metabolome analysis using capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) has the potential to improve coverage of metabolite detection because of its high selectivity and sensitivity. Configuration of the interface between CE and HRMS to meet the ground connection is essential for enabling independent regulation of the electrical currents in the CE and electrospray field. In the present study, we applied an electrospray-ionization adapter equipped with a grounded nebulizer to CE-HRMS and tested the analytical performance for 34 charged compounds. The extracted-ion electropherograms, consisting of seven sets of isomers, showed reasonable peak shapes and separation for the annotation of each metabolite. The levels of 34 target analytes in a standard mixture were determined with a dynamic range of at least 10, maintaining linearity with r > 0.9. The repeatability and intermediate precision above the lower limit of quantification showed the relative standard deviation to be lower than 20%. In the spike-recovery experiment, 27 of the 34 metabolites in plasma extract were recovered at a rate of 80 to 120%, suggesting high accuracy. Furthermore, we assessed the feasibility of our platform in metabolome analysis using human-plasma extract. The results showed successful detection of 270 metabolites, indicating the potential of our platform to yield higher coverage of the metabolome. In addition, analysis of dilution integrity demonstrated the quantitative ability of metabolome analysis with CE-HRMS, although the existence of saturation or matrix effects were seen in the case of 33 of the metabolites. This study indicates that our platform has great potential for large-scale metabolome analysis of plasma for biological studies and clinical biomarker screening.

Article	Year
Metabolomics-based approach for ranking the candidate structures of unidentified peaks in capillary electrophoresis time-of-flight mass spectrometry. Electrophoresis\| PubMed ID: 27943356	2017
Plasma metabolome analysis of patients with major depressive disorder. Psychiatry and clinical neurosciences\| PubMed ID: 29356314	2018
Metabolomics Platform with Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Plasma Analysis. Analytical chemistry\| PubMed ID: 30500154	2019

Kazunori Sasaki

Biography

JoVE Journal Publications

Other Publications