Evaluation of the High-Field Orbitrap Fusion for Compound Annotation in Metabolomics

Abstract

Annotation of signals of interest represents a key point in mass spectrometry-based metabolomics studies. The first level of investigation is the elemental composition, which can be deduced from accurately measured masses and isotope patterns. However, accuracy of these two parameters remains to be evaluated on last generation mass spectrometers to determine the level of confidence that can be used during the annotation process. In this context, we evaluated the performance of the Orbitrap Fusion mass spectrometer for the first time and demonstrated huge potential for metabolite annotation via elemental composition determination. This work was performed using a set of 50 standard compounds analyzed under LC/MS conditions in solvent and biological media. Accurate control of the number of trapped ions proved mandatory to avoid space charge effects, ensure sub-ppm mass accuracy (using external calibration), and reliable measurement of isotopic patterns at 500,000 resolution. On the basis of the results, we propose standard optimized experimental conditions for performing robust and accurate untargeted metabolomics on the Orbitrap Fusion at high mass measurement and mass spectral accuracy.

Comparative Evaluation of Data Dependent and Data Independent Acquisition Workflows Implemented on an Orbitrap Fusion for Untargeted Metabolomics

“…In this context, a set of 47 standard molecules was first used to develop and optimize DDA and DIA (SWATH) acquisition methods, involving either or both non-resonant higher energy collision-induced dissociation (HCD), or ion trap resonant collision-induced dissociation (CID) conditions. The 47 chemicals comprising this test set were those already used in a previous study [6] and well ionized in ESI+ mode while exhibiting large chemical, mass and retention time diversity when analyzed on a C 18 column (Supplementary Material, Table S1). As we observed in a previous study [29], such a C 18 column is not the most suited one to separate metabolite isomers, but is the most demanding in terms of MS-MS/MS acquisition speed (due to the narrow chromatographic peaks).…”

“…We also observed that complex and composite DIA-MS/MS spectra can be efficiently exploited to identify metabolites in plasma thanks to a reference tandem spectral library made from authentic standards while also providing a valuable data resource for further identification of unknown metabolites. Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.Metabolites 2020, 10, 158 2 of 17 and thus, metabolite annotation and identification still represent major bottlenecks in untargeted metabolomics [3,4].The successful identification of metabolites requires a combination of information lines exploited in conjunction, including accurate LC retention time, high mass measurement accuracy (<1 ppm), isotope pattern and isotope fine structure measurement as well as high-quality fragmentation spectra [5,6]. High mass resolution (>100,000) allows for the measurement of isotope pattern and isotope fine structure (through the distinction of isobaric isotopes), which is particularly useful for confident compound annotation [6].…”

“…Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.Metabolites 2020, 10, 158 2 of 17 and thus, metabolite annotation and identification still represent major bottlenecks in untargeted metabolomics [3,4].The successful identification of metabolites requires a combination of information lines exploited in conjunction, including accurate LC retention time, high mass measurement accuracy (<1 ppm), isotope pattern and isotope fine structure measurement as well as high-quality fragmentation spectra [5,6]. High mass resolution (>100,000) allows for the measurement of isotope pattern and isotope fine structure (through the distinction of isobaric isotopes), which is particularly useful for confident compound annotation [6]. One of the most valuable elements to confirm metabolite annotation or reduce the list of possible annotations is the acquisition of fragmentation spectra and their comparison to reference MS/MS spectra included in spectral libraries [7].Experimental approaches to acquire MS/MS data are generally referred to as Data Dependent or Data Independent Acquisition (DDA and DIA, respectively).…”

“…The successful identification of metabolites requires a combination of information lines exploited in conjunction, including accurate LC retention time, high mass measurement accuracy (<1 ppm), isotope pattern and isotope fine structure measurement as well as high-quality fragmentation spectra [5,6]. High mass resolution (>100,000) allows for the measurement of isotope pattern and isotope fine structure (through the distinction of isobaric isotopes), which is particularly useful for confident compound annotation [6]. One of the most valuable elements to confirm metabolite annotation or reduce the list of possible annotations is the acquisition of fragmentation spectra and their comparison to reference MS/MS spectra included in spectral libraries [7].…”

Proposal for a chemically consistent way to annotate ions arising from the analysis of reference compounds under ESI conditions: A prerequisite to proper mass spectral database constitution in metabolomics

“…Two categories of isotopologues may be distinguished: (a) the endogenous ones, ie, species that exist due to the natural occurrence of different isotopes for a chemical element (such as carbon‐13, nitrogen‐15, or sulfur‐34 that are natural isotopes of their most common counterpart carbon‐12, nitrogen‐14, and sulfur‐32, with relative abundance of 1.11%, 0.38%, and 4.40%, respectively); (b) the isotopically labelled ones, ie, species isotopically enriched with less abundant naturally occurring isotopes (eg, carbon‐13, hydrogen‐2, or nitrogen‐15) and synthetically produced either via biological or chemical processes. Endogenous isotopologues. Today, high‐resolution mass spectrometry and especially the new Orbitrap instrument generation (reaching a resolution of 500 000 at m/z 200) can allow distinguishing between isobaric isotopologue ions co‐occurring in the natural isotope pattern of a given metabolite, which can be really helpful for an accurate metabolite annotation . For instance, the isobaric 15 N 1 ‐ and 13 C 1 ‐isotopologues of the same molecule might be potentially resolved thanks to HRMS.…”