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

Teresa Mairinger,Stephan Hann

doi:10.1007/s00216-017-0339-1

Abstract

A novel analytical approach based on liquid chromatography coupled to quadrupole time of flight mass spectrometry, employing data-dependent triggering for analysis of isotopologue and tandem mass isotopomer fractions of metabolites of the primary carbon metabolism was developed. The implemented QTOFMS method employs automated MS/MS triggering of higher abundant, biologically relevant isotopologues for generating positional information of the respective metabolite. Using this advanced isotopologue selective fragmentation approach enables the generation of significant tandem mass isotopomer data within a short cycle time without compromising sensitivity. Due to a lack of suitable reference material certified for isotopologue ratios, a Pichia pastoris cell extract with a defined 13C distribution as well as a cell extract from a 13C-based metabolic flux experiment were employed for proof of concept. Moreover, a method inter-comparison with an already established GC-CI-(Q)TOFMS approach was conducted. Both methods showed good agreement on isotopologue and tandem mass isotopomer distributions for the two different cell extracts.Graphical abstractSchematic overview of data-dependent isotopologue fragmentation for acquisition of isotopologue and tandem mass isotopomer fractions

Highlights

For the characterization of alteration within metabolic networks, the introduction of stable isotope tracers into an organism and the consecutive analysis of the resulting stable isotope-labeling patterns of metabolites has become a key technology to accurately estimate intracellular metabolic rates, i.e., fluxes [1,2,3,4,5]
One of the approaches is to generate product ion spectra, known as parallel reaction monitoring (PRM) mode, where the precursor ion is filtered in the first quadrupole and fragmented in the collision cell, and all generated product ions are monitored in MS2 scans on a high-resolution, accurate mass spectrometer [15].This PRM approach using high mass accuracy mass spectrometry was first employed in the field of quantitative targeted proteomics [15]
For the proof of concept, citric acid was used, as for this metabolite GC-QTOFMS and LC-QTOFMS analysis result in the same fragment ion regarding the number of carbons in the backbone

Summary

Introduction

For the characterization of alteration within metabolic networks, the introduction of stable isotope tracers into an organism and the consecutive analysis of the resulting stable isotope-labeling patterns of metabolites has become a key technology to accurately estimate intracellular metabolic rates, i.e., fluxes [1,2,3,4,5]. This isotopic enrichment of intracellular metabolites, typically on the basis of 13C labeling, is mostly analyzed on mass spectrometric detectors [6].

Methods

Results

Conclusion