70
sufficient coverage over a chromatographic peak, producing quantitative, reproducible data for each metabolite.
Untargeted metabolomics comes with a different set of requirements due to the
chemical diversity of analytes that are characterized in a single analysis. Here, an
instrument’s ability to quickly and efficiently identify a molecule which may be of
interest for further investigation by MS/MS is critical. There exist two main methods of addressing this problem: data-dependent-acquisition (DDA) and dataindependent- acquisition (DIA). Various vendors have different names for these
processes, but in general these two techniques prevail in untargeted metabolomics
workflows [9]. DDA has proven to be effective in the field of proteomics for years
[60]. For DDA, tandem mass spectra are collected for m/z values selected from a
previously acquired MS spectrum; where the instrument is set to perform an initial
MS scan, determine the N most abundant ions, and isolate those for MS/MS analysis [61]. This entire process must take place in milliseconds [62], and is repeated
several times over the course of a peak for effective and reliable MS/MS data.
DIA has grown more prominent recently, where independent of the MS data, the
instrument indiscriminately isolates mass windows (which in some cases can be
chosen by the operator) across the entire MS mass range [63]. Each of these windows is sequentially isolated for fragmentation, meaning that ions within each window will proceed to the collision cell, producing fragments that are subsequently
analyzed [64]. One exception to this generalization is in Waters platforms, which
refer to their version of DIA as ‘MS
E’
and fragments all precursor ions simultaneously following an initial MS scan. Regardless of the how the DIA is carried out, as
with DDA, this entire process must be repeated several times within a peak width
for reliable fragmentation and quantitation [64]. The difficulty in this approach is
pairing fragment ions with their parent ion counterparts. This requires sophisticated
software programs for data annotation [65]. Thus, the specific data analysis approach
must be carefully considered before acquiring data to ensure success.
It should be noted that fragmentation, while integral to both workflows, serves a
slightly different purpose for targeted and untargeted metabolomics. In a targeted
experiment, fragmentation is used as validation of the previously known identity of
a given molecule. The resulting fragment is measured, meaning that quantitation is
based solely on the abundance of fragment ions [66]. In untargeted metabolomics,
MS/MS is used to extract more information about the possible identity of a given
molecule [11]. In this case, any quantitative measurements are most frequently
made using MS data [11]. Lastly, the mass resolving power of the instrument tends
to differ between targeted and untargeted workflows. Untargeted metabolomics
benefits from higher mass resolving power as exact mass measurements in the MS
data can aid in identifying a metabolite [67]. For this reason, Orbitrap-based and
QTOF systems have prevailed as the ideal platforms for an untargeted experiment.
Orbitraps offer varying levels of mass resolving power, ranging from 15,000–240,000
[68], and can be modulated by the operator to suit an experiment. While not as high
performance, QTOF instruments offer mass resolving powers of up to 60,000 on
current platforms [69]. In contrast, a targeted experiment relies on specific fragmentation and retention time for identification of a metabolite, rather than mass
E. S. Rivera et al.
sufficient coverage over a chromatographic peak, producing quantitative, reproducible data for each metabolite.
Untargeted metabolomics comes with a different set of requirements due to the
chemical diversity of analytes that are characterized in a single analysis. Here, an
instrument’s ability to quickly and efficiently identify a molecule which may be of
interest for further investigation by MS/MS is critical. There exist two main methods of addressing this problem: data-dependent-acquisition (DDA) and dataindependent- acquisition (DIA). Various vendors have different names for these
processes, but in general these two techniques prevail in untargeted metabolomics
workflows [9]. DDA has proven to be effective in the field of proteomics for years
[60]. For DDA, tandem mass spectra are collected for m/z values selected from a
previously acquired MS spectrum; where the instrument is set to perform an initial
MS scan, determine the N most abundant ions, and isolate those for MS/MS analysis [61]. This entire process must take place in milliseconds [62], and is repeated
several times over the course of a peak for effective and reliable MS/MS data.
DIA has grown more prominent recently, where independent of the MS data, the
instrument indiscriminately isolates mass windows (which in some cases can be
chosen by the operator) across the entire MS mass range [63]. Each of these windows is sequentially isolated for fragmentation, meaning that ions within each window will proceed to the collision cell, producing fragments that are subsequently
analyzed [64]. One exception to this generalization is in Waters platforms, which
refer to their version of DIA as ‘MS
E’
and fragments all precursor ions simultaneously following an initial MS scan. Regardless of the how the DIA is carried out, as
with DDA, this entire process must be repeated several times within a peak width
for reliable fragmentation and quantitation [64]. The difficulty in this approach is
pairing fragment ions with their parent ion counterparts. This requires sophisticated
software programs for data annotation [65]. Thus, the specific data analysis approach
must be carefully considered before acquiring data to ensure success.
It should be noted that fragmentation, while integral to both workflows, serves a
slightly different purpose for targeted and untargeted metabolomics. In a targeted
experiment, fragmentation is used as validation of the previously known identity of
a given molecule. The resulting fragment is measured, meaning that quantitation is
based solely on the abundance of fragment ions [66]. In untargeted metabolomics,
MS/MS is used to extract more information about the possible identity of a given
molecule [11]. In this case, any quantitative measurements are most frequently
made using MS data [11]. Lastly, the mass resolving power of the instrument tends
to differ between targeted and untargeted workflows. Untargeted metabolomics
benefits from higher mass resolving power as exact mass measurements in the MS
data can aid in identifying a metabolite [67]. For this reason, Orbitrap-based and
QTOF systems have prevailed as the ideal platforms for an untargeted experiment.
Orbitraps offer varying levels of mass resolving power, ranging from 15,000–240,000
[68], and can be modulated by the operator to suit an experiment. While not as high
performance, QTOF instruments offer mass resolving powers of up to 60,000 on
current platforms [69]. In contrast, a targeted experiment relies on specific fragmentation and retention time for identification of a metabolite, rather than mass
E. S. Rivera et al.