TY - CHAP
T1 - Analysis of Cell Metabolism Using LC-MS and Isotope Tracers
AU - MacKay, Gillian M.
AU - Zheng, Liang
AU - Van Den Broek, Niels J.F.
AU - Gottlieb, Eyal
N1 - Publisher Copyright:
© 2015 Elsevier Inc. All rights reserved.
PY - 2015/9/9
Y1 - 2015/9/9
N2 - Here we discuss our methods to analyze small polar compounds involved in central carbon metabolism using LC-MS. Methods described include sample extraction procedures for cells and medium, as well as for plasma/serum, urine, CSF, and tissue samples. Different extraction solvents are assessed. Our methods for using 13C stable isotope tracers to examine the kinetics and distributions of mass isotopologues of many metabolites are discussed. Quantification methods are described for 13C stable isotope tracer experiments as well as for unlabeled experiments. These methods were applied in a fumarate hydratase deficient cell model to show how isotope tracing can demonstrate shifts in metabolic pathways and, together with metabolite exchange rates, can be used to gain insights into changes in cell metabolism.
AB - Here we discuss our methods to analyze small polar compounds involved in central carbon metabolism using LC-MS. Methods described include sample extraction procedures for cells and medium, as well as for plasma/serum, urine, CSF, and tissue samples. Different extraction solvents are assessed. Our methods for using 13C stable isotope tracers to examine the kinetics and distributions of mass isotopologues of many metabolites are discussed. Quantification methods are described for 13C stable isotope tracer experiments as well as for unlabeled experiments. These methods were applied in a fumarate hydratase deficient cell model to show how isotope tracing can demonstrate shifts in metabolic pathways and, together with metabolite exchange rates, can be used to gain insights into changes in cell metabolism.
KW - Exactive and Q-Exactive mass spectrometry
KW - Fumarate hydratase
KW - HILIC chromatography
KW - LC-MS
KW - Metabolite extraction
KW - Metabolite quantification
KW - Stable isotope tracers
UR - http://www.scopus.com/inward/record.url?scp=84941025166&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84941025166&partnerID=8YFLogxK
U2 - 10.1016/bs.mie.2015.05.016
DO - 10.1016/bs.mie.2015.05.016
M3 - Chapter
C2 - 26358905
AN - SCOPUS:84941025166
T3 - Methods in Enzymology
SP - 171
EP - 196
BT - Methods in Enzymology
PB - Academic Press Inc.
ER -