ORCID Profile
0000-0002-0762-3492
Current Organisations
University of Natural Resources and Life Sciences
,
Kyushu University
,
University of Massachusetts Amherst
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Publisher: Wiley
Date: 08-2017
Abstract: Reversed-phase LC combined with high-resolution mass spectrometry (HRMS) is one of the most popular methods for cellular metabolomics studies. Due to the difficulties in analyzing a wide range of polarities encountered in the metabolome, 100%-wettable reversed-phase materials are frequently used to maximize metabolome coverage within a single analysis. Packed with silica-based sub-3 μm diameter particles, these columns allow high separation efficiency and offer a reasonable compromise for metabolome coverage within a single analysis. While direct performance comparison can be made using classical chromatographic characterization approaches, a comprehensive assessment of the column's performance for cellular metabolomics requires use of a full LC-HRMS workflow in order to reflect realistic study conditions used for cellular metabolomics. In this study, a comparison of several reversed-phase LC columns for metabolome analysis using such a dedicated workflow is presented. All columns were tested under the same analytical conditions on an LC-TOF-MS platform using a variety of authentic metabolite standards and biotechnologically relevant yeast cell extracts. Data on total workflow performance including retention behavior, peak capacity, coverage, and molecular feature extraction repeatability from these columns are presented with consideration for both nontargeted screening and differential metabolomics workflows using authentic standards and Pichia pastoris cell extract s les.
Publisher: Elsevier BV
Date: 12-2019
DOI: 10.1016/J.TALANTA.2019.120147
Abstract: In this study, we present a novel selective cleanup/enrichment method based on metal oxide solid phase extraction combined with quantitative gas chromatography-tandem mass spectrometry and ion exchange chromatography-tandem mass spectrometry for the analysis of phosphorylated metabolites in yeast cell extracts relevant to biotechnological processes. Following screening of several commercially available metal oxide-based enrichment materials, all steps of the enrichment process (loading, washing and elution) were optimized for both the selective enrichment of 12 phosphorylated compounds from the glycolysis and pentose phosphate pathways, and the simultaneous removal of highly abundant matrix components such as organic acids and sugars. The full analytical workflow was then validated to meet the demands of accurate quantification of phosphorylated metabolites in yeast (Pichia pastoris) cell extracts using the best performing material and cleanup/enrichment method combined with quantification strategies based on internal standardization with isotopically labeled internal standards and external calibration. A good recovery (>70%) for 5 of the 12 targeted phosphorylated compounds with RSDs of less than 6.0% was obtained while many sugars, organic acids and amino acids were removed (>99% of glucose, and >95% of aspartate, succinate, glutamate, alanine, glycine, serine, threonine, proline, and valine). The use of isotopically labeled internal standards added to the s les prior to SPE, enables accurate quantification of the metabolites as it compensates for errors introduced during s le pretreatment and GC-MS or LC-MS analysis. To the best of our knowledge, this is the first time an effective and selective metal oxide-based affinity chromatography cleanup/enrichment method was designed and applied successfully for intracellular phosphorylated metabolites.
Publisher: Springer Science and Business Media LLC
Date: 13-07-2019
DOI: 10.1007/S00216-019-02021-8
Abstract: This study of ion accumulation/release behavior relevant to ion mobility-mass spectrometry (IM-MS) as employed for non-targeted metabolomics involves insight from theoretical studies, and controlled reference experiments involving measurement of low and high molecular mass metabolites in varying concentrations within a complex matrix (yeast extracts). Instrumental settings influencing ion trapping (accumulation time) and release conditions in standard and multiplexed operation have been examined, and translation of these insights to liquid chromatography (LC) in combination with drift tube IM-MS measurements has been made. The focus of the application is non-targeted metabolomics using carefully selected s les to allow quantitative interpretations to be made. Experimental investigation of the IM-MS ion utilization efficiency particularly focusing on the use of the Hadamard transform multiplexing with 4-bit pseudo-random pulsing sequence for assessment of low and high molecular mass metabolites is compared with theoretical modeling of gas-phase behavior of small and large molecules in the IM trapping funnel. Increasing the trapping time for small metabolites with standard IM-MS operation is demonstrated to have a deleterious effect on maintaining a quantitative representation of the metabolite abundance. The application of these insights to real-world non-targeted metabolomics assessment of intracellular extracts from biotechnologically relevant production processes is presented, and the results were compared to LC×IM-MS measurements of the same s les. Spiking of a uniformly
Location: United States of America
No related grants have been discovered for Si-Hung Le.