title: Determination of amino acids in colon cancer cells by using UHPLC-MS/MS and [U-13C5]-glutamine as the isotope tracer.
abstract: Rapid and simple quantitative analysis of intracellular metabolites is a critical tool for monitoring the alteration of biologically significant metabolites in cell lines or in vivo. We established an ultra-high performance liquid chromatography (UHPLC) method, equipped with hydrophilic interaction liquid chromatography (HILIC) column coupled to tandem mass spectrometry (MS/MS) for the simultaneous determination of 19 amino acids and 2 related derivatives in human cell lines. Chromatographic separation was achieved within 20min using a BEH amide column, with aqueous mobile phase containing 20mM ammonium acetate and 20mM ammonium hydroxide, and acetonitrile as the organic mobile phase. Amino acids were analyzed in positive ion multiple reaction monitoring (MRM) mode without the need of derivatization. Intra- and inter-day precisions were less than 13.7%. The method was successfully applied to simultaneously detect the 21 compounds in a human colon cancer cell line DLD1. Moreover, metabolite fate of glutamine-derived carbons into amino acids in DLD1 cells was successfully traced by using [U-13C5] glutamine as the isotope tracer. Metabolic consequences of glutaminolysis inhibition on amino acid metabolism were evaluated. Analysis of12C- and U-13C-labeled amino acids revealed the significantly decreased incorporation of [U-13C5]-glutamine derived carbons into aspartate, alanine and ornithine, indicating impaired metabolic flux via the tricarboxylic acid cycle and the urea cycle.
authors.affiliation: State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
authors.forename: Xiaona
authors.initials: X
authors.lastname: Li
authors.affiliation: Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Chi Chun
authors.initials: CC
authors.lastname: Wong
authors.affiliation: State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Tang
authors.affiliation: State Key Laboratory for Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau, China.
authors.forename: Jianlin
authors.initials: J
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
authors.forename: Shangfu
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Gastroenterology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China, Institute of Gastroenterology, Zhejiang University, Hangzhou, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Qian
authors.affiliation: Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Jiaying
authors.initials: J
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
authors.forename: Zhiyi
authors.initials: Z
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China. Electronic address: zwcai@hkbu.edu.hk.
authors.forename: Zongwei
authors.initials: Z
authors.lastname: Cai
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D002247:Carbon Isotopes; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D011849:Radioactive Tracers; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D011849:Radioactive Tracers; D005973:Glutamine
keywords: (13)C(5)-glutamine isotope tracer; Amino acids; Glutaminolysis-inhibition; Hydrophilic interaction liquid chromatography; Mass spectrometry