Simultaneous quantification of metabolites involved in central carbon and energy metabolism using reversed-phase liquid chromatography-mass spectrometry and in vitro 13C labeling

Wen Chu Yang, Miroslav Sedlak, Fred E. Regnier, Nathan Mosier, Nancy Ho, Jiri Adamec

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylics acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a 13C-labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R2 > 0.9975. The detection limits at a signalto-noise ratio of 3 were below 1.0 μM (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were identified and quantified from yeast lysate with a relative standard deviation of less than 10%.

Original languageEnglish (US)
Pages (from-to)9508-9516
Number of pages9
JournalAnalytical chemistry
Volume80
Issue number24
DOIs
StatePublished - Dec 15 2008

Fingerprint

Liquid chromatography
Metabolites
Labeling
Mass spectrometry
Carbon
Pentoses
Sugar Acids
Glucose-6-Phosphate
Coenzymes
Carboxylic Acids
Isomers
Yeast
Nucleotides
Phosphates
Energy Metabolism
Calibration
Acids

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Simultaneous quantification of metabolites involved in central carbon and energy metabolism using reversed-phase liquid chromatography-mass spectrometry and in vitro 13C labeling. / Yang, Wen Chu; Sedlak, Miroslav; Regnier, Fred E.; Mosier, Nathan; Ho, Nancy; Adamec, Jiri.

In: Analytical chemistry, Vol. 80, No. 24, 15.12.2008, p. 9508-9516.

Research output: Contribution to journalArticle

@article{d6d44bdadcb44d06af88ec1282c21475,
title = "Simultaneous quantification of metabolites involved in central carbon and energy metabolism using reversed-phase liquid chromatography-mass spectrometry and in vitro 13C labeling",
abstract = "Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylics acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a 13C-labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R2 > 0.9975. The detection limits at a signalto-noise ratio of 3 were below 1.0 μM (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were identified and quantified from yeast lysate with a relative standard deviation of less than 10{\%}.",
author = "Yang, {Wen Chu} and Miroslav Sedlak and Regnier, {Fred E.} and Nathan Mosier and Nancy Ho and Jiri Adamec",
year = "2008",
month = "12",
day = "15",
doi = "10.1021/ac801693c",
language = "English (US)",
volume = "80",
pages = "9508--9516",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "24",

}

TY - JOUR

T1 - Simultaneous quantification of metabolites involved in central carbon and energy metabolism using reversed-phase liquid chromatography-mass spectrometry and in vitro 13C labeling

AU - Yang, Wen Chu

AU - Sedlak, Miroslav

AU - Regnier, Fred E.

AU - Mosier, Nathan

AU - Ho, Nancy

AU - Adamec, Jiri

PY - 2008/12/15

Y1 - 2008/12/15

N2 - Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylics acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a 13C-labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R2 > 0.9975. The detection limits at a signalto-noise ratio of 3 were below 1.0 μM (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were identified and quantified from yeast lysate with a relative standard deviation of less than 10%.

AB - Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylics acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a 13C-labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R2 > 0.9975. The detection limits at a signalto-noise ratio of 3 were below 1.0 μM (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were identified and quantified from yeast lysate with a relative standard deviation of less than 10%.

UR - http://www.scopus.com/inward/record.url?scp=58149141938&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=58149141938&partnerID=8YFLogxK

U2 - 10.1021/ac801693c

DO - 10.1021/ac801693c

M3 - Article

C2 - 19007244

AN - SCOPUS:58149141938

VL - 80

SP - 9508

EP - 9516

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 24

ER -