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bioRxiv · 10.1101/2022.09.28.509873

Systematic analysis of in-source modifications of primary metabolites during flow-injection time-of-flight mass spectrometry

Farke, N.·Schramm, T.·Verhuelsdonk, A.·Link, H.
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Abstract

Flow-injection mass spectrometry (FI-MS) enables metabolomics studies with a very high sample-throughput. However, FI-MS is prone to in-source modifications of analytes because samples are directly injected into the electrospray ionization source of a mass spectrometer without prior chromatographic separation. Here, we spiked authentic standards of 160 primary metabolites individually into an Escherichia coli metabolite extract and measured the thus derived 160 spike-in samples by FI-MS. Our results demonstrate that FI-MS can capture a wide range of chemically divers analytes within 30 seconds measurement time. However, the data also revealed extensive in-source modifications. Across all 160 spike-in samples, we identified significant increases of 11,013 ion peaks in positive and negative mode combined. To explain these unknown m/z features, we connected them to the m/z feature of the (de-)protonated metabolite using information about mass differences and MS2 spectra. This resulted in networks that explained on average 49 % of all significant features. The networks showed that a single metabolite undergoes compound specific and often sequential in-source modifications like adductions, chemical reactions, and fragmentations. Our results show that FI-MS generates complex MS1 spectra, which leads to an overestimation of significant features, but neutral losses and MS2 spectra explain many of these features. HighlightsO_LIFI-MS enables measurements of chemically divers metabolites. C_LIO_LIExtensive in-source modifications during electrospray ionization are detected by FI-MS. C_LIO_LIA network approach explains 49 % of all recorded in-source modifications. C_LI

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Farke, N., Schramm, T., Verhuelsdonk, A., Link, H.. 2022-09-28. Systematic analysis of in-source modifications of primary metabolites during flow-injection time-of-flight mass spectrometry. https://doi.org/10.1101/2022.09.28.509873

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