Resolving isobaric interferences in direct infusion tandem mass spectrometry

Abstract

Rationale The co‐fragmentation of precursors in direct infusion (DI) tandem high‐resolution mass spectrometry (HRMS) can complicate the fragment spectra and consequently lead to false hits during compound identification. Methods The method herein described, termed IQAROS (incremental quadrupole acquisition to resolve overlapping spectra), modulates the intensities of precursors and fragments by stepwise movement of the quadrupole isolation window over the mass‐to‐charge (m/z) range of the precursors. The modulated signals are then deconvoluted by a linear regression model to reconstruct the fragment spectra with less interference. The hardware to demonstrate the use of IQAROS was an orbitrap with electrospray ionization (ESI) or secondary electrospray ionization (SESI), although the method can also be applied to other ionization techniques or mass analyzers. Results Assessing the performance of IQAROS with isobaric standards revealed that the reconstructed fragment spectra match with spectra acquired from the pure standards and that more compounds were correctly identified compared with the classical approach with the quadrupole centered at the m/z value of the precursor of interest. Moreover, the strength of IQAROS is exemplified by the identification of two isobaric biomarkers directly from a breath sample with SESI‐HRMS. Conclusions With IQAROS, cleaner fragment spectra of co‐fragmenting isobars during DI‐HRMS analysis can be obtained. IQAROS can easily be set up by the standard graphical user interface of the instrument. Therefore, it facilitates the characterization of features of interest in samples analyzed by DI‐HRMS, for example, in high‐throughput or real‐time metabolomics.

Breath response following a nutritional challenge monitored by secondary electrospray ionization high-resolution mass spectrometry

“…reaching the collision cell and consequently leading to chimeric fragment spectra, a recently developed methodology improving real-time MS 2 -experiments called IQAROS was employed [30]. After processing the data, the reconstructed fragment spectra were processed within SIRIUS [31] to obtain structural candidates for each feature with help of the [32] and [33] database.…”

“…If a feature had a maximum intensity over the threshold (50% of the intensity within the window) direct fragmentation with the same settings as in section 2.3 and collision energies of 10, 35 and 50 was attempted. For features below the threshold, an incremental quadrupole acquisition for resolving spectra (IQAROS) sequence was set up [30]. Briefly, scans were set up with the same collision energies and a mass resolution of 17 500.…”

Identification of Exhaled Metabolites in Children with Cystic Fibrosis

“…However, the use of on-line MS 2 spectra and literature matching for structure annotation has some limitations. (1) The recording of on-line MS 2 spectra often leads to co-fragmentation of several precursors within the mass isolation window of 0.7 ± 0.1 Da, which complicates the correct an-notation based on fragment spectra analysis [17]. (2) We did not record fragmentation spectra by LC-MS or GC-MS to confirm the putative compound structure.…”

“…In general, compound identification is a crucial but complex and limiting step of metabolomic studies [ 16 ]. Chemical structure annotation in on-line breath analysis is challenging, due to the lack of a separation step and co-fragmentation of compounds within the quadrupole isolation window, which complicates spectral matching [ 17 ]. Methods for compound identification that have been previously applied in SESI-HRMS studies include the sampling of exhaled breath condensate (EBC) for analysis using liquid chromatography mass spectrometry (LC-MS) and subsequent fragment spectra and/or retention time matching to standard chemicals [ 10 , 13 ], yielding the highest degree of identification certainty.…”

Non-invasive monitoring of microbiota and host metabolism using Secondary electrospray ionization-Mass spectrometry

“…SESI sources are commercially available and can be interfaced with commercial high-resolution mass spectrometers [27, 28, 56]. The data generated from SESI-MS is compatible with standard software for direct-injection MS-based metabolomics [57, 58], and online MS2 fragmentation for compound identification can be performed [59, 60]. Development is still needed for collecting and processing time-series data, since most current MS-based metabolomics datasets have low time-resolution or do not consider time-dependent changes at all.…”