Dataset Information

Quantitative multiple fragment monitoring with enhanced in-source fragmentation/annotation mass spectrometry.

ABSTRACT: Analytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold standard in clinical chemistry. However, tandem mass spectrometers come at high capital expenditure and maintenance costs. We recently showed that it is possible to generate very similar results using a much simpler single mass spectrometry detector by performing enhanced in-source fragmentation/annotation (EISA) combined with correlated ion monitoring. Here we provide a step-by-step protocol for optimizing the analytical conditions for EISA, so anyone properly trained in liquid chromatography-mass spectrometry can follow and apply this technique for any given analyte. We exemplify the approach by using 2-hydroxyglutarate (2-HG) which is a clinically relevant metabolite whose D-enantiomer is considered an 'oncometabolite', characteristic of cancers associated with mutated isocitrate dehydrogenases 1 or 2 (IDH1/2). We include procedures for determining quantitative robustness, and show results of these relating to the analysis of DL-2-hydroxyglutarate in cells, as well as in serum samples from patients with acute myeloid leukemia that contain the IDH1/2 mutation. This EISA-mass spectrometry protocol is a broadly applicable and low-cost approach for the quantification of small molecules that has been developed to work well for both single-quadrupole and time-of-flight mass analyzers.

SUBMITTER: Bernardo-Bermejo S

PROVIDER: S-EPMC10364092 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Quantitative multiple fragment monitoring with enhanced in-source fragmentation/annotation mass spectrometry.

Bernardo-Bermejo Samuel S Xue Jingchuan J Hoang Linh L Billings Elizabeth E Webb Bill B Honders M Willy MW Venneker Sanne S Heijs Bram B Castro-Puyana María M Marina María Luisa ML van den Akker Erik B EB Griffioen Marieke M Siuzdak Gary G Giera Martin M Sánchez-López Elena E

Nature protocols 20230208 4

Analytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold standard in clinical chemistry. However, tandem mass spectrometers come at high capital expenditure and maintenance costs. We recently showed that it is possible to generate very similar results using a much simpler single mass spectrometry detector by performing enhanced in-source fragmentation/annotatio ...[more]

PMID: 36755131

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Project description:Electrospray ionization (ESI) in-source fragmentation (ISF) has traditionally been minimized to promote precursor molecular ion formation, and therefore its value in molecular identification is underappreciated. In-source annotation algorithms have been shown to increase confidence in putative identifications by using ubiquitous in-source fragments. However, these in-source annotation algorithms are limited by ESI sources that are generally designed to minimize ISF. In this study, enhanced in-source fragmentation annotation (eISA) was created by tuning the ISF conditions to generate in-source fragmentation patterns comparable with higher energy fragments generated at higher collision energies as deposited in the METLIN MS/MS library, without compromising the intensity of precursor ions (median loss ≤10% in both positive and negative ionization modes). The analysis of 50 molecules was used to validate the approach in comparison to MS/MS spectra produced via data dependent acquisition (DDA) and data independent acquisition (DIA) mode with quadrupole time-of-flight mass spectrometry (QTOF-MS). Enhanced ISF as compared to QTOF DDA enabled higher peak intensities for the precursor ions (median: 18 times in negative mode and 210 times in positive mode), with the eISA fragmentation patterns consistent with METLIN for over 90% of the molecules with respect to fragment relative intensity and m/z. eISA also provides higher peak intensity as opposed to QTOF DIA for over 60% of the precursor ions in negative mode (median increase: 20%) and for 88% of the precursor ions in positive mode (median increase: 80%). Molecular identification with eISA was also successfully validated from the analysis of a metabolic extract from macrophages. An interesting side benefit of enhanced ISF is that it significantly improved molecular identification confidence with low resolution single quadrupole mass-spectrometry-based untargeted LC/MS experiments. Overall, enhanced ISF allowed for eISA to be used as a more sensitive alternative to other QTOF DIA and DDA approaches, and further, it enabled the acquisition of ESI TOF and ESI single quadrupole mass spectrometry instrumentation spectra with improved molecular identification confidence.

Project description:Enhanced in-source fragmentation/annotation (EISA) has recently been shown to produce fragment ions that match tandem mass spectrometry data across a wide range of small molecules. EISA has been developed to facilitate data-dependent acquisition (DDA), data-independent acquisiton (DIA), and multiple-reaction monitoring (MRM), enabling molecular identifications in untargeted metabolomics and targeted quantitative single-quadrupole MRM (Q-MRM) analyses. Here, EISA has been applied to peptide-based proteomic analysis using optimized in-source fragmentation to generate fragmentation patterns for a mixture of 38 peptides, which were comparable to the b- and y-type fragment ions typically observed in tandem MS experiments. The optimal in-source fragmentation conditions at which high-abundance peptide fragments and precursor ions coexist were compared with automated data-dependent acquisition (DDA) in the same quadrupole time-of-flight (QTOF-MS) mass spectrometer, generating a significantly higher fragment percentage of peptides from both singly and doubly charged b- and y-type fragment (b+, y+, b2+, and y2+) ions. Higher fragment percentages were also observed for these fragment ion series over linear ion trap instrumentation. An XCMS-EISA annotation/deconvolution program was developed, making use of the retention time and peak shape continuity between precursor fragment ions, to perform automated proteomic data analysis on the enhanced in-source fragments. Post-translational modification (PTM) characterization on peptides was demonstrated with EISA, producing fragment ions corresponding to a neutral loss of phosphoric acid with greater intensity than observed with DDA on a QTOF-MS. Moreover, Q-MRM demonstrated the ability to use EISA for peptide quantification. The availability of more sophisticated in-source fragmentation informatics, beyond XCMS-EISA, will further enable EISA for sensitive autonomous identification and Q-MRM quantitative analyses in proteomics.

Dataset Information

Quantitative multiple fragment monitoring with enhanced in-source fragmentation/annotation mass spectrometry.

Publications

Quantitative multiple fragment monitoring with enhanced in-source fragmentation/annotation mass spectrometry.

Similar Datasets

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