The first data-processing
step of AllExtract and TracExtract detects pairs of native and labeled
metabolite ion signals. For each data file, the following steps are
successively performed for all recorded LC-HRMS scans. Each mass peak
is initially considered to represent the monoisotopic ion M of a native
metabolite or biotransformation product. This assumption is verified
with the following criteria:
(i) An isotopologue M′ from
the labeled metabolite or biotransformation product must be present
in the same MS scan. As the charge (z) and the number
of labeling isotopes (Xn) cannot be deduced
from the single mass peak M, MetExtract II tests several user-defined
combinations of Xn and z. For each combination, an m/z value
for the putatively labeled isotopologue M′ is calculated [m/z(M′) = m/z(M) + (XnΔm)/z] and searched for in the same MS scan. If a
mass peak with such an m/z value
is present within a user-defined tolerance window (i.e., intrascan
mass accuracy of the HRMS instrument used), it is considered a putative
labeled signal M′. Together with Xn and z, M and M′ represent a putative ion
pair. However, if no mass peak is found for any combination of Xn and z values, the current
mass peak M is rejected.
(ii) The observed abundances of both
mass peaks M and M′
must exceed a certain, user-defined intensity threshold. If any do
not, the ion pair is rejected.
(iii) Depending on the experimental
setup, the user can optionally
define an intensity ratio of M:M′ (e.g., the ratio of native
to labeled tracer applied in the biological experiment). If the ratio
is not within the specified tolerance window, the ion pair is rejected.
(iv) The observed isotopologue patterns originating from native
and labeled metabolite ions must match with their respective theoretical
patterns. This is tested by comparing the observed isotopologue ratios
by using the intensity ratio of M + 1 to M [I(M +
1)/I(M)] as well as M′ – 1 to M′
[I(M′ – 1)/I(M′)]
with the expected ratios for a compound having the assigned number
of labeling isotopes (Xn) as well as the
isotopic abundance with either the principal isotope or the labeling
isotope. Theeoretical ratios for such isotopologues are calculated
by use ofeq 1 with a = Xn, s =
1, and e = relative abundance of the principal isotope
in nature (e.g., for 12C, 98.93%) or the isotopic enrichment
with the labeling isotope (the 13C isotopic enrichment
used).
Labeled
biotransformation products may partly consist of native
(nonlabeled) structure units (any conjugated moiety from the native
biological system), which do not contribute to the m/z shift between M and M′. These moieties
need to be accounted for in the native ion forms by the TracExtract
module when the observed isotopologue ratio I(M +
1)/I(M) is compared to the theoretical ratio for
Xn labeling atoms as their presence increases
the theoretical ratio I(M + 1)/I(M). Thus, the observed ratio I(M + 1)/I(M) is corrected for the ratio of nonlabeled atoms I(M′ + 1)/I(M′) (corresponding to all
atoms of the labeling element in the native moiety but not in the
tracer itself) before it is tested against its corresponding theoretical
ratio. This corrected ratio I(M + 1)/I(M) – I(M′ + 1)/I(M′) represents only the number of atoms of the labeling-element
originating from the studied tracer. The ratio I(M′
– 1)/I(M′), however, is derived solely
from the Xn labeling isotope atoms and
must not be calculated differently than in the AllExtract module.
If the observed and calculated theoretical isotopologue ratios deviate
by less than a user-defined tolerance window (the expected relative
isotopologue abundance error of the used HRMS instrument), the ion
pair is accepted. If either of the two isotopologue ratio tests exceeds
the maximum allowed tolerance, the ion pair is rejected. Any MS signal
pair passing these verification criteria is considered to be an ion
of a native and a corresponding labeled metabolite or biotransformation
product.
step of AllExtract and TracExtract detects pairs of native and labeled
metabolite ion signals. For each data file, the following steps are
successively performed for all recorded LC-HRMS scans. Each mass peak
is initially considered to represent the monoisotopic ion M of a native
metabolite or biotransformation product. This assumption is verified
with the following criteria:
(i) An isotopologue M′ from
the labeled metabolite or biotransformation product must be present
in the same MS scan. As the charge (z) and the number
of labeling isotopes (Xn) cannot be deduced
from the single mass peak M, MetExtract II tests several user-defined
combinations of Xn and z. For each combination, an m/z value
for the putatively labeled isotopologue M′ is calculated [m/z(M′) = m/z(M) + (XnΔm)/z] and searched for in the same MS scan. If a
mass peak with such an m/z value
is present within a user-defined tolerance window (i.e., intrascan
mass accuracy of the HRMS instrument used), it is considered a putative
labeled signal M′. Together with Xn and z, M and M′ represent a putative ion
pair. However, if no mass peak is found for any combination of Xn and z values, the current
mass peak M is rejected.
(ii) The observed abundances of both
mass peaks M and M′
must exceed a certain, user-defined intensity threshold. If any do
not, the ion pair is rejected.
(iii) Depending on the experimental
setup, the user can optionally
define an intensity ratio of M:M′ (e.g., the ratio of native
to labeled tracer applied in the biological experiment). If the ratio
is not within the specified tolerance window, the ion pair is rejected.
(iv) The observed isotopologue patterns originating from native
and labeled metabolite ions must match with their respective theoretical
patterns. This is tested by comparing the observed isotopologue ratios
by using the intensity ratio of M + 1 to M [I(M +
1)/I(M)] as well as M′ – 1 to M′
[I(M′ – 1)/I(M′)]
with the expected ratios for a compound having the assigned number
of labeling isotopes (Xn) as well as the
isotopic abundance with either the principal isotope or the labeling
isotope. Theeoretical ratios for such isotopologues are calculated
by use of
1, and e = relative abundance of the principal isotope
in nature (e.g., for 12C, 98.93%) or the isotopic enrichment
with the labeling isotope (the 13C isotopic enrichment
used).
Labeled
biotransformation products may partly consist of native
(nonlabeled) structure units (any conjugated moiety from the native
biological system), which do not contribute to the m/z shift between M and M′. These moieties
need to be accounted for in the native ion forms by the TracExtract
module when the observed isotopologue ratio I(M +
1)/I(M) is compared to the theoretical ratio for
Xn labeling atoms as their presence increases
the theoretical ratio I(M + 1)/I(M). Thus, the observed ratio I(M + 1)/I(M) is corrected for the ratio of nonlabeled atoms I(M′ + 1)/I(M′) (corresponding to all
atoms of the labeling element in the native moiety but not in the
tracer itself) before it is tested against its corresponding theoretical
ratio. This corrected ratio I(M + 1)/I(M) – I(M′ + 1)/I(M′) represents only the number of atoms of the labeling-element
originating from the studied tracer. The ratio I(M′
– 1)/I(M′), however, is derived solely
from the Xn labeling isotope atoms and
must not be calculated differently than in the AllExtract module.
If the observed and calculated theoretical isotopologue ratios deviate
by less than a user-defined tolerance window (the expected relative
isotopologue abundance error of the used HRMS instrument), the ion
pair is accepted. If either of the two isotopologue ratio tests exceeds
the maximum allowed tolerance, the ion pair is rejected. Any MS signal
pair passing these verification criteria is considered to be an ion
of a native and a corresponding labeled metabolite or biotransformation
product.
