{"title":"通过异构体选择性精确靶向亲水相互作用液相色谱-串联质谱分析法全面覆盖糖酵解和五糖磷酸代谢途径","authors":"Kristian Serafimov, Michael Lämmerhofer","doi":"10.1021/acs.analchem.4c03490","DOIUrl":null,"url":null,"abstract":"The accurate liquid chromatography-tandem mass spectrometry analysis of phosphorylated isomers from glycolysis and pentose phosphate pathways is a challenging analytical problem in metabolomics due to extraction problems from the biological matrix, adherence to stainless steel surfaces leading to tailing in LC, and incomplete separation of hexose and pentose phosphate isomers. In this study, we present a targeted HILIC-ESI-MS/MS method based on a BEH amide fully porous 1.7 μm particle column with an inert surface coating of column hardware and multiple reaction monitoring (MRM) acquisition fully covering the glycolysis and pentose phosphate pathway metabolites. To minimize contact of the phosphorylated analytes with stainless steel surfaces, a μ-ESI-MS probe with a hybrid electrode made of PEEKsil was employed. Optimized HILIC gradient elution conditions with 100 mM ammonium formate (pH 11) provided the separation of hexose monophosphate and pentose phosphate isomers. To ensure good retention time repeatability in HILIC, perfluoroalkoxy alkane bottles were used for the mobile phase (with sd over 60 runs between 0.01 and 0.02 min). For the quantitative assay, the U-<sup>13</sup>C-labeled cell extract was spiked prior to extraction by metal oxide-based affinity chromatography (MOAC) with TiO<sub>2</sub> beads. The concentrations of the 24 targets were quantified in HeLa and human embryonic kidney (HEK293) cells. Erastin-induced ferroptosis in HEK293 cells was accompanied by enhanced levels of fructose-1,6-bis-phosphate, 2- and 3-phosphoglycerate, and 2,3-bis-phosphoglycerate.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7000,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Comprehensive Coverage of Glycolysis and Pentose Phosphate Metabolic Pathways by Isomer-Selective Accurate Targeted Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Assay\",\"authors\":\"Kristian Serafimov, Michael Lämmerhofer\",\"doi\":\"10.1021/acs.analchem.4c03490\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"The accurate liquid chromatography-tandem mass spectrometry analysis of phosphorylated isomers from glycolysis and pentose phosphate pathways is a challenging analytical problem in metabolomics due to extraction problems from the biological matrix, adherence to stainless steel surfaces leading to tailing in LC, and incomplete separation of hexose and pentose phosphate isomers. In this study, we present a targeted HILIC-ESI-MS/MS method based on a BEH amide fully porous 1.7 μm particle column with an inert surface coating of column hardware and multiple reaction monitoring (MRM) acquisition fully covering the glycolysis and pentose phosphate pathway metabolites. To minimize contact of the phosphorylated analytes with stainless steel surfaces, a μ-ESI-MS probe with a hybrid electrode made of PEEKsil was employed. Optimized HILIC gradient elution conditions with 100 mM ammonium formate (pH 11) provided the separation of hexose monophosphate and pentose phosphate isomers. To ensure good retention time repeatability in HILIC, perfluoroalkoxy alkane bottles were used for the mobile phase (with sd over 60 runs between 0.01 and 0.02 min). For the quantitative assay, the U-<sup>13</sup>C-labeled cell extract was spiked prior to extraction by metal oxide-based affinity chromatography (MOAC) with TiO<sub>2</sub> beads. The concentrations of the 24 targets were quantified in HeLa and human embryonic kidney (HEK293) cells. Erastin-induced ferroptosis in HEK293 cells was accompanied by enhanced levels of fructose-1,6-bis-phosphate, 2- and 3-phosphoglycerate, and 2,3-bis-phosphoglycerate.\",\"PeriodicalId\":27,\"journal\":{\"name\":\"Analytical Chemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.7000,\"publicationDate\":\"2024-10-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1021/acs.analchem.4c03490\",\"RegionNum\":1,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1021/acs.analchem.4c03490","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
Comprehensive Coverage of Glycolysis and Pentose Phosphate Metabolic Pathways by Isomer-Selective Accurate Targeted Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Assay
The accurate liquid chromatography-tandem mass spectrometry analysis of phosphorylated isomers from glycolysis and pentose phosphate pathways is a challenging analytical problem in metabolomics due to extraction problems from the biological matrix, adherence to stainless steel surfaces leading to tailing in LC, and incomplete separation of hexose and pentose phosphate isomers. In this study, we present a targeted HILIC-ESI-MS/MS method based on a BEH amide fully porous 1.7 μm particle column with an inert surface coating of column hardware and multiple reaction monitoring (MRM) acquisition fully covering the glycolysis and pentose phosphate pathway metabolites. To minimize contact of the phosphorylated analytes with stainless steel surfaces, a μ-ESI-MS probe with a hybrid electrode made of PEEKsil was employed. Optimized HILIC gradient elution conditions with 100 mM ammonium formate (pH 11) provided the separation of hexose monophosphate and pentose phosphate isomers. To ensure good retention time repeatability in HILIC, perfluoroalkoxy alkane bottles were used for the mobile phase (with sd over 60 runs between 0.01 and 0.02 min). For the quantitative assay, the U-13C-labeled cell extract was spiked prior to extraction by metal oxide-based affinity chromatography (MOAC) with TiO2 beads. The concentrations of the 24 targets were quantified in HeLa and human embryonic kidney (HEK293) cells. Erastin-induced ferroptosis in HEK293 cells was accompanied by enhanced levels of fructose-1,6-bis-phosphate, 2- and 3-phosphoglycerate, and 2,3-bis-phosphoglycerate.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.