We report a segmented spectrum scan method using Orbitrap
MS in
chemical isotope labeling (CIL) liquid chromatography–mass
spectrometry (LC–MS) for improving the metabolite detection
efficiency. In this method, the full m/z range is divided into multiple segments with the scanning of each
segment to produce multiple narrow-range spectra during the LC data
acquisition. These segmented spectra are separately processed to extract
the peak pair information with each peak pair arising from a differentially
labeled metabolite in the analysis of a mixture of 13C
and 12C reagent-labeled samples. The sublists of peak pairs
are merged to form the final peak pair list from the LC–MS
run. Various experimental conditions, including automatic gain control
(AGC) values, mass resolutions, segment m/z widths, number of segments, and total data acquisition
time in the LC run, were examined to arrive at an optimal setting
in the segment scan for increasing the number of detectable metabolites
while maintaining the same analysis time as in the full scan. The
optimal method used a segment width of 120 m/z with 60k resolution for a 16 min CIL LC–MS run.
Using dansyl-labeled human urine samples as an example, we demonstrated
that this method could detect 5867 peak pairs or metabolites (not
features), compared to 3765 peak pairs detectable in a full scan,
representing a 56% gain. Out of 5867 peak pairs, 5575 (95.0%) could
be identified or mass-matched. The relative quantification accuracy
was slightly reduced (81% peak pairs were within ±25% of the
expected peak ratio of 1.0 in full, compared to 87% in the full scan)
due to the inclusion of more low-abundance peak pairs in the segment
scan. The peak ratio measurement precision was not significantly affected
by the segment scan. We also showed the increase of the peak pair
number detectable from 3843 in the full scan to 7273 (89% gain) using
the Orbitrap operated at 120k resolution with a 60 m/z segment width when multiple repeat sample injections
were used. Thus, segment scan Orbitrap MS is an enabling method for
detecting coeluting metabolites in CIL LC–MS for increasing
the metabolomic coverage.
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