Abstract
BackgroundMetabolic flux analysis has become an established method in systems biology and functional genomics. The most common approach for determining intracellular metabolic fluxes is to utilize mass spectrometry in combination with stable isotope labeling experiments. However, before the mass spectrometric data can be used it has to be corrected for biases caused by naturally occurring stable isotopes, by the analytical technique(s) employed, or by the biological sample itself. Finally the MS data and the labeling information it contains have to be assembled into a data format usable by flux analysis software (of which several dedicated packages exist). Currently the processing of mass spectrometric data is time-consuming and error-prone requiring peak by peak cut-and-paste analysis and manual curation. In order to facilitate high-throughput metabolic flux analysis, the automation of multiple steps in the analytical workflow is necessary.ResultsHere we describe iMS2Flux, software developed to automate, standardize and connect the data flow between mass spectrometric measurements and flux analysis programs. This tool streamlines the transfer of data from extraction via correction tools to 13C-Flux software by processing MS data from stable isotope labeling experiments. It allows the correction of large and heterogeneous MS datasets for the presence of naturally occurring stable isotopes, initial biomass and several mass spectrometry effects. Before and after data correction, several checks can be performed to ensure accurate data. The corrected data may be returned in a variety of formats including those used by metabolic flux analysis software such as 13CFLUX, OpenFLUX and 13CFLUX2.ConclusioniMS2Flux is a versatile, easy to use tool for the automated processing of mass spectrometric data containing isotope labeling information. It represents the core framework for a standardized workflow and data processing. Due to its flexibility it facilitates the inclusion of different experimental datasets and thus can contribute to the expansion of flux analysis applications.
Highlights
Metabolic flux analysis has become an established method in systems biology and functional genomics
The determination of the amount of label taken up is complicated by several factors: (i) naturally occurring stable isotopes (NOIs) of almost all elements found in metabolites, including 13C: 1.1%, 2H 0.0115%, 17O 0.038%, 18O 0.2% 15N 0.366%, and 34S: 4.2%, [6,7]; (ii) additional elements with stable isotopes introduced by derivatization such as 29Si or 30Si, natural abundance 4.7% and 3.1%, respectively [8,9,10,11]; (iii) proton gain or loss during mass spectrometric analysis
Results and discussion iMS2Flux has been designed to act as a high-throughput framework for mass spectrometric (MS) data analysis, targeting metabolic flux analysis (MFA) as its primary application, but is not inherently limited to MFA
Summary
Metabolic flux analysis has become an established method in systems biology and functional genomics. A range of useful software is available that perform different aspects required for MFA including MS data extraction [30,31], data correction [11,32], model development and analysis [33,34], see Table 1. We describe iMS2Flux, a tool that provides a framework for a standardized workflow to automatically process MS data from isotope tracer experiments.
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