Enhanced in-Source Fragmentation Annotation Enables Novel Data Independent Acquisition and Autonomous METLIN Molecular Identification

Abstract

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.

A Metabolomics Workflow for Analyzing Complex Biological Samples Using a Combined Method of Untargeted and Target-List Based Approaches

“…It offers a graphical user interface (GUI) and has built-in multiple previews to control and modify setting for peak integration. The compound identification is based on a user-defined database with m / z values with retention times and can be connected directly to several online resources, e.g., PubChem [ 33 ], HMDB [ 34 ], or METLIN [ 35 , 36 ]. In our study, we used Compound Discoverer 3.0 (Thermo), a commercial software tool, XCMS online, and Lipid Data Analyzer 2.6, both open-source tools.…”

“…Chemspider, which integrates BioCyc [ 33 ], Human Metabolome Database [ 34 ], and KEGG database [ 35 ], was used to annotate features based on exact mass with a mass tolerance of 5 ppm as well as the CD internal database (an endogenous metabolites database of 4400 compounds). The data set obtained was used to generate a list of potential targets taking into account the level of confidence in annotations implemented by the Metabolomics Standards Initiative (MSI) of the Metabolomics Society ( Table 1 ) [ 14 , 36 ]. In detail, the confidence level of annotation was created as follows: In the first step, all hits from the mzCloud—MS/MS scoring database—greater than 80% score were exported.…”

Metabolite discovery through global annotation of untargeted metabolomics data

“…Additional abiotic edge types LC-MS metabolomics may include additional abiotic relationships. In orbitrap data, these include oligomers, multi-charge species, heterodimers, in-source fragments of known or unknown metabolites 36 , and ringing artifact peaks surrounding high intensity ions 20,37 . These relationships were Database MS2 of known metabolites can be used to identify known ion fragmentation peaks 36 .…”

“…In orbitrap data, these include oligomers, multi-charge species, heterodimers, in-source fragments of known or unknown metabolites 36 , and ringing artifact peaks surrounding high intensity ions 20,37 . These relationships were Database MS2 of known metabolites can be used to identify known ion fragmentation peaks 36 . If candidate annotation of node v is annotated with a HMDB ID associated with database MS2 spectrum, and m/z of node u matches to a fragment m/z in 's MS2 spectrum, then a database fragment edge will connect such two nodes.…”

Single Quadrupole Multiple Reaction Monitoring Q-MRM enables Quantitative Mass Spectrometry

“…Two separate technologies: 1) soft atmospheric pressure ionization with enhanced insource fragmentation/annotation (EISA) and 2) an MRM approach that is collectively called EISA-MRM, have been combined to provide a quantitative analysis technology in a single quadrupole MS system in which QqQ-MRM is not available (Figure 1). EISA is a recently introduced high energy electrospray ionization in-source fragmentation/annotation approach that promotes the generation of both molecular ions and their respective fragments at high abundance 11,12 . EISA contrasts to traditional electrospray as a soft ionization technology and is proving to be advantageous since the resulting fragment ions are typically the same as those generated in tandem mass spectrometry experiments 11,12 .…”