69
up-stream of a mass spectrometer, providing a second level of separation as well as
affording higher peak capacity [9]. Ion mobility is not without shortcomings however, especially that the addition of ion mobility in a metabolomics experiment has
been shown to reduce overall sensitivity [53].
4.4 Mass Spectrometry
In metabolomics, the instrument of choice is dependent on the experiment being
conducted, where different mass spectrometer platforms are ideal for different types
of assays. For targeted experiments, an instrument capable of interrogating many
known molecules on a time-scale that is compatible with the chromatographic timescale is critical. Targeted assays are commonly quantitative; therefore, it is important that the instrument selected has good quantitative capabilities and sensitivity.
Usually this type of work is done by a triple-quadrupole or QTRAP system [15].
Untargeted experiments have different needs, as unknown molecules must be
selected for fragmentation in a manner that permits broad coverage of the analytes.
Orbitrap-based and quadrupole time-of-flight (QTOF) systems have proven themselves optimal for these types of workflows [6, 55]. There are many other types of
mass spectrometers which can be used for LC-MS-based metabolomics, but this
chapter will focus specifically on these as they are the platforms that play a central
role in the field.
Triple quadrupole and QTRAP platforms are the dominant mass spectrometers
used in the field of targeted metabolomics [55]. These instruments are very similar
in design, sharing an electrospray source followed by optics for the transmission of
ions to an initial quadrupole capable of isolating specific m/z windows. A second
quadrupole is then used as a collision cell for collision-induced dissociation (CID)
to fragment precursor ions for MS/MS analysis [56]. These two instruments differ
in their final stage where a triple quadrupole is equipped with a third quadrupole
used to isolate a particular m/z of the fragments created in the collision cell for
transmission to the detector. The QTRAP is equipped with a ion trap rather than a
conventional quadrupole, which is capable of not only of performing subsequent
fragmentation events on product ions but also accumulating ions for increased sensitivity [57]. This instrument can also be operated as a conventional triple quadrupole instrument. Both systems are capable of isolating a precursor ion, fragmenting,
and monitoring the presence of specific fragments on the order of milliseconds [58].
The monitoring of a specific fragment of a specific parent ion is referred to as a
selected reaction monitoring (SRM), and this approach can be multiplexed so that
multiple analytes can be monitored in the same assay with high specificity. This
approach is referred to as multiple reaction monitoring (MRM), and is paramount
for targeted LC-MS techniques [59]. Practically, these mass spectrometers are capable of quantitatively monitoring upwards of 50 unique transitions within milliseconds [58]. This level of speed allows for dozens of metabolites to be measured with
4 Fundamentals of Mass Spectrometry-Based Metabolomics
up-stream of a mass spectrometer, providing a second level of separation as well as
affording higher peak capacity [9]. Ion mobility is not without shortcomings however, especially that the addition of ion mobility in a metabolomics experiment has
been shown to reduce overall sensitivity [53].
4.4 Mass Spectrometry
In metabolomics, the instrument of choice is dependent on the experiment being
conducted, where different mass spectrometer platforms are ideal for different types
of assays. For targeted experiments, an instrument capable of interrogating many
known molecules on a time-scale that is compatible with the chromatographic timescale is critical. Targeted assays are commonly quantitative; therefore, it is important that the instrument selected has good quantitative capabilities and sensitivity.
Usually this type of work is done by a triple-quadrupole or QTRAP system [15].
Untargeted experiments have different needs, as unknown molecules must be
selected for fragmentation in a manner that permits broad coverage of the analytes.
Orbitrap-based and quadrupole time-of-flight (QTOF) systems have proven themselves optimal for these types of workflows [6, 55]. There are many other types of
mass spectrometers which can be used for LC-MS-based metabolomics, but this
chapter will focus specifically on these as they are the platforms that play a central
role in the field.
Triple quadrupole and QTRAP platforms are the dominant mass spectrometers
used in the field of targeted metabolomics [55]. These instruments are very similar
in design, sharing an electrospray source followed by optics for the transmission of
ions to an initial quadrupole capable of isolating specific m/z windows. A second
quadrupole is then used as a collision cell for collision-induced dissociation (CID)
to fragment precursor ions for MS/MS analysis [56]. These two instruments differ
in their final stage where a triple quadrupole is equipped with a third quadrupole
used to isolate a particular m/z of the fragments created in the collision cell for
transmission to the detector. The QTRAP is equipped with a ion trap rather than a
conventional quadrupole, which is capable of not only of performing subsequent
fragmentation events on product ions but also accumulating ions for increased sensitivity [57]. This instrument can also be operated as a conventional triple quadrupole instrument. Both systems are capable of isolating a precursor ion, fragmenting,
and monitoring the presence of specific fragments on the order of milliseconds [58].
The monitoring of a specific fragment of a specific parent ion is referred to as a
selected reaction monitoring (SRM), and this approach can be multiplexed so that
multiple analytes can be monitored in the same assay with high specificity. This
approach is referred to as multiple reaction monitoring (MRM), and is paramount
for targeted LC-MS techniques [59]. Practically, these mass spectrometers are capable of quantitatively monitoring upwards of 50 unique transitions within milliseconds [58]. This level of speed allows for dozens of metabolites to be measured with
4 Fundamentals of Mass Spectrometry-Based Metabolomics