Related Experiment Video
Updated: Aug 14, 2026

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry
Published on: October 25, 2019
A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing
Wenyun Lu1, Elizabeth Kimball1, Joshua D Rabinowitz2
1Lewis-Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Princeton, New Jersey, USA.
Abstract:
A comprehensive method of quantifying intracellular metabolite concentrations would be a valuable addition to the arsenal of tools for holistic biochemical studies. Here, we describe a step toward the development of such method: a quantitative assay for 90 nitrogen-containing cellular metabolites. The assay involves reverse-phase high-performance liquid chromatography separation followed by electrospray ionization and detection of the resulting ions using triple-quadrupole mass spectrometry in selected reaction monitoring mode. For 79 of the 90 metabolites, the assay is linear with a limit of detection of 10 ng/mL or less. Using this method, 36 metabolites can be reliably detected in extracts of the bacterium Salmonella enterica, with the identity of each metabolite confirmed by the presence, on growing of the bacteria in (13)C-glucose, of a peak corresponding to the isotope-labeled form of the compound. Quantitation in biological samples is performed by mixing unlabeled test cell extract with (13)C-labeled standard extract, and determining the (12)C/(13)C-ratio for each metabolite. Using this approach, the metabolomes of growing (exponential phase) and carbon-starved (stationary phase) bacteria were compared, revealing 16 metabolites that are significantly down-regulated and five metabolites that are significantly up-regulated, in stationary phase.
More Related Videos
10:44Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures
Published on: March 28, 2017
08:41Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications
Published on: July 14, 2017
Related Concept Videos
Tandem Mass Spectrometry
Peptide Identification Using Tandem Mass Spectrometry
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
Mass Spectrometry: Complex Analysis
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
High-Performance Liquid Chromatography: Types of Detectors