A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways

Wellcome Open Research

article

Adhish Walvekar¹  Zeenat Rashida¹  Hemanth Maddali¹  Sunil Laxman¹ 

[1] Institute for Stem Cell biology and Regenerative Medicine;Manipal Academy of Higher Education

关键词: Mass spectrometry;    central carbon metabolism;    OBHA derivatization;    stable-isotope labeling;    labeling of amino acids and nucleotides;    kinetics of label incorporation;    metabolic flux analysis;   

DOI  :  10.12688/wellcomeopenres.14832.1

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Liquid chromatography-mass spectrometry (LC-MS/MS) based approaches are widely used for the identification and quantitation of specific metabolites, and are a preferred approach towards analyzing cellular metabolism. Most methods developed come with specific requirements such as unique columns, ion-pairing reagents and pH conditions, and typically allow measurements in a specific pathway alone. Here, we present a single column-based set of methods for simultaneous coverage of multiple pathways, primarily focusing on central carbon, amino acid, and nucleotide metabolism. We further demonstrate the use of this method for quantitative, stable isotope-based metabolic flux experiments, expanding its use beyond steady-state level measurements of metabolites. The expected kinetics of label accumulation pertinent to the pathway under study are presented with some examples. The methods discussed here are broadly applicable, minimize the need for multiple chromatographic resolution methods, and highlight how simple labeling experiments can be valuable in facilitating a comprehensive understanding of the metabolic state of cells.

【 授权许可】

CC BY