Liquid Chromatography Methods for Separation of Polar and Charged Intracellular Metabolites for 13C Metabolic Flux Analysis

Damini Jaiswal; Anjali Mittal; Deepak Nagrath; Pramod P Wangikar

doi:10.1007/978-1-0716-0159-4_3

Liquid Chromatography Methods for Separation of Polar and Charged Intracellular Metabolites for ¹³C Metabolic Flux Analysis

Methods Mol Biol. 2020:2088:33-50. doi: 10.1007/978-1-0716-0159-4_3.

Authors

Damini Jaiswal¹, Anjali Mittal², Deepak Nagrath^{2

3}, Pramod P Wangikar^{4

5

6}

Affiliations

¹ Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai, India.
² Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA.
³ Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA.
⁴ Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai, India. wangikar@iitb.ac.in.
⁵ DBT-PAN IIT Centre for Bioenergy, Indian Institute of Technology Bombay, Mumbai, India. wangikar@iitb.ac.in.
⁶ Wadhwani Research Centre for Bioengineering, Indian Institute of Technology Bombay, Mumbai, India. wangikar@iitb.ac.in.

PMID: 31893369
DOI: 10.1007/978-1-0716-0159-4_3

Abstract

Accurate quantification of mass isotopolog distribution (MID) of intracellular metabolites is a key requirement for ¹³C metabolic flux analysis (¹³C-MFA). Liquid chromatography coupled with mass spectrometry (LC/MS) has emerged as a frontrunner technique that combines two orthogonal separation strategies. While metabolomics requires separation of monoisotopic peaks, ¹³C-MFA imposes additional demands for chromatographic separation as isotopologs of metabolites significantly add to the number of analytes. In this protocol chapter, we discuss two liquid chromatography methods, namely, reverse phase ion-pairing and hydrophilic interaction chromatography (HILIC) that together can separate a wide variety of metabolites that are typically used for ¹³C metabolic flux analysis.

Keywords: HILIC; Metabolic flux analysis; Nucleotides; Reverse phase ion-pairing; Sugar phosphates.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Carbon Isotopes / chemistry*
Chromatography, Liquid / methods*
Chromatography, Reverse-Phase / methods
Metabolic Flux Analysis / methods*
Metabolome / physiology
Metabolomics / methods*
Tandem Mass Spectrometry / methods

Substances

Carbon Isotopes