blank to collect the initial exclusion list, running several blanks
is necessary to equilibrate the column and lower the background level. We run both positive mode and negative mode
to increase metabolome coverage. For positive mode, use the
full MS1 scan method to run the Acquire X blank to collect the
exclusion list. Then run the Acquire X of a pooled sample (with
mixture of equal aliquots from all the replicates and samples) to
collect the targeted inclusion list. After that, run the individual
metabolite samples with a method which includes the exclusion
list and the targeted inclusion list. After completion of the first
injection, the software will put metabolite peaks with good
fragmentation spectra into the exclusion list and keep those
without fragmentation spectra in the inclusion list. Then run an
MS
n method containing the updated exclusion list and inclusion list for the second injection (Fig. 2). This iterative process
often requires 3–5 times of replicate injections of samples, and
the more complicated metabolome will need more iterations
for deep metabolome coverage. Negative mode is run in the
same manner following the positive mode.
5. Data Analysis: Our workflow contained the following elements
nodes from ten areas.
(a) Input/Output—The Input files node has no parameters
and every processing workflow must begin with this node.
(b) Spectrum Processing—The Select Spectra node. The
Align Retention Times node performs retention time
alignment.
(c) Trace Creation—No nodes were selected.
(d) Compound Detection—The Detect Compounds node
provides unknown compound detection. The Group
Compounds node performs compound grouping across
all samples. The Fill Gaps node fills gaps across all samples,
and hides chemical background (using Blank samples).
(e) Peak Area Refinement—The Normalize Areas node
applies QC-based batch normalization if QC samples are
available. The Mark Background Compounds node.
(f) Compound Identification—The Predict Compositions
node predicts elemental compositions for all compounds.
The Search mzCloud node identifies compounds using
mzCloud from ddMS2 product spectra. This node also
performs similarity searches for all compounds with
ddMS2 data using mzCloud. The Assign Compound
Annotations node. The Search ChemSpider node identifies compounds using ChemSpider from the formula or
exact mass. The Search mzVault node.
422
Lisa David et al.
is necessary to equilibrate the column and lower the background level. We run both positive mode and negative mode
to increase metabolome coverage. For positive mode, use the
full MS1 scan method to run the Acquire X blank to collect the
exclusion list. Then run the Acquire X of a pooled sample (with
mixture of equal aliquots from all the replicates and samples) to
collect the targeted inclusion list. After that, run the individual
metabolite samples with a method which includes the exclusion
list and the targeted inclusion list. After completion of the first
injection, the software will put metabolite peaks with good
fragmentation spectra into the exclusion list and keep those
without fragmentation spectra in the inclusion list. Then run an
MS
n method containing the updated exclusion list and inclusion list for the second injection (Fig. 2). This iterative process
often requires 3–5 times of replicate injections of samples, and
the more complicated metabolome will need more iterations
for deep metabolome coverage. Negative mode is run in the
same manner following the positive mode.
5. Data Analysis: Our workflow contained the following elements
nodes from ten areas.
(a) Input/Output—The Input files node has no parameters
and every processing workflow must begin with this node.
(b) Spectrum Processing—The Select Spectra node. The
Align Retention Times node performs retention time
alignment.
(c) Trace Creation—No nodes were selected.
(d) Compound Detection—The Detect Compounds node
provides unknown compound detection. The Group
Compounds node performs compound grouping across
all samples. The Fill Gaps node fills gaps across all samples,
and hides chemical background (using Blank samples).
(e) Peak Area Refinement—The Normalize Areas node
applies QC-based batch normalization if QC samples are
available. The Mark Background Compounds node.
(f) Compound Identification—The Predict Compositions
node predicts elemental compositions for all compounds.
The Search mzCloud node identifies compounds using
mzCloud from ddMS2 product spectra. This node also
performs similarity searches for all compounds with
ddMS2 data using mzCloud. The Assign Compound
Annotations node. The Search ChemSpider node identifies compounds using ChemSpider from the formula or
exact mass. The Search mzVault node.
422
Lisa David et al.