71
resolving power [9]. A targeted metabolomics experiment is also greatly affected by
the speed of the mass spectrometer being used. For this reason, most instrument
platforms used in targeted workflows are low resolution mass spectrometers capable
of rapid scan rates that enable high-speed MRM analyses. Both targeted and untargeted workflows require high levels of data management and processing, which can
often prove to be the most cumbersome and time-intensive component of a metabolomics experiment.
4.5 Data Analysis
The validity of identifications in metabolomics studies are crucial for making biological conclusions. A useful framework for considering the confidence in the identification of a specific metabolite has been established [9]. In this framework, there
are five levels of validation ranging from a unique feature such as an accurate mass
measurement at level five (the lowest level of validation), to a validated identification which is measured against a reference standard with a confirmed structure at
level one (the highest level of validation) (Fig.4.3).
One universal challenge between targeted and untargeted experiments is determining which spectra in these large data sets are representative of a real metabolite.
In order to make confident identifications, informative features relevant to
Fig. 4.3 Levels of Confidence for Metabolite Validation. From top to bottom the least validated
level 5 to validated level 1 is shown. With increased level of validation comes increased time and
cost. A level five validated feature is a unique feature with accurate mass measurement. A level
four validation is a unique molecular formula. A level three validated feature includes tentative
structure and matches a precursor to an MS1 data based. A level two validated feature, a putative
identification matches MS/MS spectra to a database. Level two and three validations utilize
orthogonal measurements and can be techniques other than mass spectrometry such as NMR, collisional cross section, spectroscopy, or retention time. A completely validated metabolite, a level
one validation matches a metabolite to a reference standard [9]
4 Fundamentals of Mass Spectrometry-Based Metabolomics
resolving power [9]. A targeted metabolomics experiment is also greatly affected by
the speed of the mass spectrometer being used. For this reason, most instrument
platforms used in targeted workflows are low resolution mass spectrometers capable
of rapid scan rates that enable high-speed MRM analyses. Both targeted and untargeted workflows require high levels of data management and processing, which can
often prove to be the most cumbersome and time-intensive component of a metabolomics experiment.
4.5 Data Analysis
The validity of identifications in metabolomics studies are crucial for making biological conclusions. A useful framework for considering the confidence in the identification of a specific metabolite has been established [9]. In this framework, there
are five levels of validation ranging from a unique feature such as an accurate mass
measurement at level five (the lowest level of validation), to a validated identification which is measured against a reference standard with a confirmed structure at
level one (the highest level of validation) (Fig.4.3).
One universal challenge between targeted and untargeted experiments is determining which spectra in these large data sets are representative of a real metabolite.
In order to make confident identifications, informative features relevant to
Fig. 4.3 Levels of Confidence for Metabolite Validation. From top to bottom the least validated
level 5 to validated level 1 is shown. With increased level of validation comes increased time and
cost. A level five validated feature is a unique feature with accurate mass measurement. A level
four validation is a unique molecular formula. A level three validated feature includes tentative
structure and matches a precursor to an MS1 data based. A level two validated feature, a putative
identification matches MS/MS spectra to a database. Level two and three validations utilize
orthogonal measurements and can be techniques other than mass spectrometry such as NMR, collisional cross section, spectroscopy, or retention time. A completely validated metabolite, a level
one validation matches a metabolite to a reference standard [9]
4 Fundamentals of Mass Spectrometry-Based Metabolomics