Integrating MS1 and MS2 Scans in High-Resolution Parallel Reaction Monitoring Assays for Targeted Metabolite Quantification and Dynamic ¹³C-Labeling Metabolism Analysis

Abstract

Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. Initially developed for proteomic analysis, the parallel reaction monitoring (PRM) technique exploiting high-resolution MS2 fragment ion data has shown high promise for targeted metabolite quantification. Notably, MS1 ion intensity data acquired independently as part of each PRM scan cycle are often underutilized in the PRM assay. In this study, we developed an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids and shikimate pathway-related metabolites on an orthogonal QqTOF system. Concentration curve assessment revealed that exploiting both MS1 and MS2 scans in PRM analysis afforded higher sensitivity, wider dynamic range and better reproducibility than relying on either scan mode for quantification. Furthermore, Skyline was incorporated into our workflow to process the MS1/MS2 ion intensity data, and eliminate noisy signals and transitions with interferences. This integrated MS1/MS2 PRM approach was applied to targeted metabolite quantification in engineered E. coli strains for understanding of metabolic pathway modulation. In addition, this new approach, when first implemented in a dynamic C-labeling experiment, showed its unique advantage in capturing and correcting isotopomer labeling curves to facilitate nonstationaryC-labeling metabolism analysis.

Implementation of data-dependent isotopologue fragmentation in 13C-based metabolic flux analysis

“…This concept of isotopologue selective fragmentation was also implemented in a recently published study on tracking of the carbon origin of tandem MS CID fragments using liquid chromatography [ 10 ]. Another analytical method using this approach, based on liquid chromatography was just recently published by Li et al based on pure ion intensity data [ 13 ].…”

A novel phosphoinositide kinase Fab1 regulates biosynthesis of pathogenic aflatoxin in Aspergillus flavus

“…Owing to the high sensitivity and robustness, parallel reaction monitoring (PRM) has recently emerged as the "gold standard" MS technique for targeted proteomic approaches, which are routinely used for quantitative analysis of targeted proteins 47,48 . To independently validate our quantitative results, we next conducted a PRM assay for 15 of the identified DEPs.…”

Measurement of metabolic fluxes using stable isotope tracers in whole animals and human patients

“…Thus, the metabolites quantified are prespecified and by nature, this data does not allow for retrospective analysis of additional molecules. Parallel reaction monitoring partially overcomes this disadvantage by increasing the number of compounds targeted in a single analysis, but to date has been utilized only minimally in metabolomics [3,4]. …”