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Optimizing Deconvolution of Chimeric Fragmentation Spectra to reveal molecular structures of complex biological mixtures

ABSTRACT:

Direct infusion mass spectrometry (DI-MS) offers a rapid, high-throughput analysis and, compared to liquid chromatography MS (LC-MS), a different analytical window into the composition of complex biological samples. However, the lack of chromatographic separation in DI-MS results in highly complex mass spectra and 'chimeric' fragmentation spectra that simultaneously contain multiple precursors and their fragments, complicating compound identification. Recently, a data acquisition technique based on shift of isolation window was proposed: to correctly connect precursor and fragments in chimeric fragmentation spectra, multiple MS/MS spectra are recorded for the same nominal mass by stepwise shifting of the isolation window centre. The ion transmission is different in the middle of isolation window and on its edges, which causes changes in intensity of precursor ions and their fragments between MS/MS scans.

To optimize deconvolution of chimeric spectra, we recorded fragmentation spectra of 115 mixtures of isobaric compounds with masses 180, 194, 280, 302, 318 and 342 Da using direct infusion - high resolution tandem mass spectrometry. Both pairs of isobaric compounds and mixtures of up to four isobaric compounds were used. For pairs of isobaric compounds, multiple mixtures with different range of concentrations were analysed. The mixtures of compounds and solutions of pure individual compounds were prepared by diluting stock solutions in MeOH:H2O 50:50 (v./v.) solvent with addition of 0.1% (v.%) HCOOH.

The data was recorded using positive electrospray ionization, resolving power of 120 000 and higher energy collisional dissociation (HCD) fragmentation with normalized collision energy (NCE) of 35 and 65 on Orbitrap Elite. Tandem mass spectra were recorded using shifting isolating window width 1 m/z, mass ranged from (x - 0.7) to (x + 1) where x is nominal mass of [M+H]+ molecular ions of isobaric compounds, with 0.02 m/z step between consecutive fragmentation events. The mass difference between isobaric compounds ranged from 0.0034 to 0.0905 m/z (mass deviations range from 11 to 500 ppm). Additionally, fragmentation spectra of individual compounds were recorded using the same NCE and isolation window width. Finally, the blank fragmentation spectra of nominal masses corresponding to nominal masses of isobaric compounds were recorded using the same solvent with 1% (v.%) addition of DMSO, same set of NCE and 3 m/z wide isolation window.

INSTRUMENT(S): Direct infusion MS - positive

PROVIDER: MTBLS13066 | MetaboLights | 2025-09-29

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS13066_DI-MS_positive__metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS13066_DI-MS_positive__metabolite_profiling_v2_maf.tsv	Tabular
	s_MTBLS13066.txt	Txt

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Optimizing Deconvolution of Chimeric Fragmentation Spectra to reveal molecular structures of complex biological mixtures

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