manually integrated pattern trace peaks were automatically linked to detected
compounds from targeted and untargeted mechanisms.
Introduction
14C
labeling is used extensively to trace the path of biochemical reactions in
metabolism or biomarker studies. Although LC/HRMS techniques are commonly
employed for these studies, labeled compound profiling in complex biological samples
remains a challenge due to factors such as complex matrixes and insufficient
resolution.
Relative Abundance
This study demonstrates a simple yet powerful labeled compound detection and
profiling workflow using the very high resolution Orbitrap Fusion mass spectrometer
and Compound Discoverer software.
2
3
Caroline Ding,1 Tim Stratton,1 Hans Pfaff,2 Hans Grensemann,Parent
Ji MaCompound:
1
compound
is AmgenGermany;
proprietary.
Thermo Fisher Scientific, San Jose, CA, USA; 2Thermo FisherThe
Scientifi
c, Bremen,
3
Formula: C26H29N7O2 with one carbon replaced with 14C  14CC25H29N7O2
Amgen Inc, South San Francisco, CA, USA
Poster Note 64497
Radio-Labeled Compound Detection
Using Isotopic Structures From Very High
Resolution Mass Spectrometry
Methods
Radio-Labeled Compound Detection
Using Isotopic Structures Fr
Co
F:
2, Ji Ma3
Caroline Ding1, Tim Stratton1, Hans Pfaff2,Monoisotopic
Hans mass:
Grensemann
473.24152
Preparation
1. Thermo Fisher Scientific, San Jose, CA,Sample
USA
2. Thermo Fisher Scientific, Bremen
Overview
Purpose: Confident detection and profiling of metabolites with effective matrix
background removal by employing 14C labeling and utilizing very high resolution mass
spectrometry in one single workflow.
The sample was dosed and incubated in RLM with NADPH at a concentration of 1uM.
The sample was quenched with 3 volumes of methanol containing 3% formic acid and
collected at T0hr and T1hr. After centrifugation, the supernatant was subjected to LCMS analysis.
FIGURE 1. Workflow tree in Compound Discoverer software which includes
Liquid
PatternChromatography
Tracer node to create a trace for 14C compounds, Expected Finder node to
detect targeted
transformation compounds
and
Unknown
Detector
node
to detect
Samples
were chromatographically
separated by
a gradient
using
an Agilent
1290
untargeted
UPLC
and a compounds.
CAPCELL PAK IF column (2X100mm, 2um).
Mass Spectrometry
Methods: The parent compound in study is fully labeled with one 14C. Samples were
prepared by incubating with RLM and NAPDH and collected at T0hr and T1hr time
points. HRMS full scan followed by data dependent data were collected on the Thermo
ScientificTM Orbitrap FusionTM TribridTM mass spectrometer with 240k and 120k
resolution respectively. Data analysis was done within Thermo ScientificTM Compound
DiscovererTM 1.0 software using one single processing workflow. The workflow
employed the Pattern Tracer node to extract out chromatographic traces from both time
points representing 14C containing compounds, the Expected Finder node for targeted
1
1 compound
compound detection, and the Unknown
Detector node for untargeted
detection.
The HRAM analysis was conducted on an Orbitrap Fusion mass spectrometer
equipped with a HESI NG ion source. Full scan MS data were collected at resolving
powers of 240K and data dependent at 120K.
removed matrix background and revealed 14C containing compounds. Comparison of
pattern traces between the two time points helped eliminate impurity compounds.
Linking compound detection with the pattern traces to get m/z, isotope pattern and
spectrum were nicely done by manual peak integration on the pattern trace. The
manually integrated pattern trace peaks were automatically linked to detected
compounds from targeted and untargeted mechanisms.
relevant peak information.
Radio-Labeled Compound Detection Using Isotopic Structures Fr
Data Analysis
The HRAM full scan data was processed by Compound Discoverer software using a
single processing workflow (Figure 1).
patterns from parent compound2(Figure 2) were3used instead of
Caroline Ding , Tim Stratton , Hans Pfaff2,Experimental
Hansenrichment
Grensemann
Ji Comparison
Ma of three different
theoretical
ratios to achieve better ,
results.
patterns used by the Pattern Trace node were evaluated (Figure 3) in order to select
1.
Thermo
Fisher
Scientific,
San
Jose, CA,theUSA
2. most
Thermo
Fisher
Bremen
Results:
The Pattern Trace
node in Compound
Discoverer software
effectively
best pattern that
effectively reduces
background,Scientific,
in the mean time, retains
Overview
Purpose: Confident detection and profiling of metabolites with effective matrix
background removal by employing 14C labeling and utilizing very high resolution mass
spectrometry in one single workflow.
14C labeling is used extensively to trace the path of biochemical reactions in
Methods:
The
parent compound
study is LC/HRMS
fully labeled
with one 14
C. commonly
Samples were
metabolism
or biomarker
studies.inAlthough
techniques
are
prepared
with RLM
andcompound
NAPDH and
collected
at T0hr biological
and T1hr samples
time
employedby
forincubating
these studies,
labeled
profiling
in complex
points.
HRMS
full scan
followed
by data
dependent
data
were and
collected
on the Thermo
remains
a
challenge
due
to
factors
such
as
complex
matrixes
insufficient
TM
TM
TM
Scientific
resolution. Orbitrap Fusion Tribrid mass spectrometer with 240k and 120k
resolution respectively. Data analysis was done within Thermo ScientificTM Compound
This study TM
demonstrates
simple
yetsingle
powerful
labeled compound
detection
and
Discoverer
1.0 softwareausing
one
processing
workflow. The
workflow
profiling workflow
using
the very
high
resolution
Fusion mass
spectrometer
employed
the Pattern
Tracer
node
to extract
out Orbitrap
chromatographic
traces
from both time
14C containing
and Compound
Discoverer
software.
compounds, the Expected Finder node for targeted
points
representing
compound detection, and the Unknown Detector node for untargeted compound
detection.
Methods
Relative Abundance
Introduction
Results: The Pattern Trace node in Compound Discoverer software effectively
Parent Compound:
removed
matrix background and revealed 14C containing compounds. Comparison of
pattern
traces between
theproprietary.
two time points helped eliminate impurity compounds.
The compound
is Amgen
Linking compound detection with the pattern traces to get m/z, isotope pattern and
14CC H N O
Formula:
C
H
N
O
with
one
carbon peak
replaced
with 14Conthe
26 29
7 2 done by manual
25 29 trace.
7 2 The
spectrum were
nicely
integration
pattern
manually
integrated
trace peaks were automatically linked to detected
Monoisotopic
mass:pattern
473.24152
compounds from targeted and untargeted mechanisms.
Sample Preparation
The sample was dosed and incubated in RLM with NADPH at a concentration of 1uM.
Introduction
The sample was quenched with 3 volumes of methanol containing 3% formic acid and
T0hr
and
T1hr. Aftertocentrifugation,
supernatant reactions
was subjected
to LCis
used
extensively
trace the paththe
of biochemical
in
MS analysis.or biomarker studies. Although LC/HRMS techniques are commonly
metabolism
employed
for these studies, labeled compound profiling in complex biological samples
Liquid Chromatography
remains a challenge due to factors such as complex matrixes and insufficient
Samples
were
chromatographically separated by a gradient using an Agilent 1290
resolution.
UPLC and a CAPCELL PAK IF column (2X100mm, 2um).
This study demonstrates a simple yet powerful labeled compound detection and
Mass Spectrometry
profiling
workflow using the very high resolution Orbitrap Fusion mass spectrometer
and
Discoverer
software.on an Orbitrap Fusion mass spectrometer
The Compound
HRAM analysis
was conducted
Relative Abundance
14
collected
at
C labeling
equipped with a HESI NG ion source. Full scan MS data were collected at resolving
powers of 240K and data dependent at 120K.
Methods
Data Analysis
Parent
Compound:
The compound
HRAM full scan
data proprietary.
was processed by Compound Discoverer software using a
The
is Amgen
single processing workflow (Figure 1).
Formula: C26H29N7O2 with one carbon replaced with 14C  14CC25H29N7O2
Experimental patterns from parent compound (Figure 2) were used instead of
Monoisotopic
mass: 473.24152
theoretical enrichment
ratios to achieve better results. Comparison of three different
patterns Preparation
used by the Pattern Trace node were evaluated (Figure 3) in order to select
Sample
the best pattern that most effectively reduces background, in the mean time, retains
The
sample
was
dosed and incubated in RLM with NADPH at a concentration of 1uM.
relevant
peak
information.
The sample was quenched with 3 volumes of methanol containing 3% formic acid and
collected at T0hr and T1hr. After centrifugation, the supernatant was subjected to LC-
FIGURE 1. Workflow tree in Compound Discoverer software which includes
Pattern Tracer node to create a trace for 14C compounds, Expected Finder node to
detect targeted transformation compounds and Unknown Detector node to detect
untargeted compounds.
FIGURE 2. Raw full ms spectrum showing full ms pattern of parent compound
from 14C labeling.
Compd_B_RLM_NADPH_t1h_240k #1714 RT: 17.86 AV: 1 NL: 8.98E6
F: FTMS + p ESI Full ms [150.0000-1000.0000]
100
A2
474.24908
90
A3
80
70
60
A4
A0
50
40
475.25128
30
20
10
0
471.48642
471.5
472.24686
472.0
472.71927
472.5
473.25024
473.0
473.5
474.11301
474.0
m/z
474.37082
474.88409
474.5
476.25522
475.0
475.5
476.0
476.5
FIGURE 3. Isotope Ratio Editor in Pattern Trace node showing input of
experimental custom pattern.
Three different patterns were evaluated
FIGURE 2. Raw full ms spectrum showing full ms pattern of parent compound
1. Pattern consisting A0 and A2 only
from 14C labeling.
Compd_B_RLM_NADPH_t1h_240k #1714 RT: 17.86 AV: 1 NL: 8.98E6
F: FTMS + p ESI Full ms [150.0000-1000.0000]
100
A2
474.24908
2. Pattern consisting A0, A2 and A3
90
A3
80
70
60
A4
A0
50
40
475.25128
30
20
10
0
471.48642
471.5
472.24686
472.0
472.71927
472.5
473.25024
473.0
473.5
474.11301
474.0
m/z
3.474.37082
Pattern consisting A0, A476.25522
2, A3 and A4
474.88409
474.5
475.0
475.5
476.0
476.5
FIGURE 3. Isotope Ratio Editor in Pattern Trace node showing input of
experimental custom pattern.
Three different patterns were evaluated
FIGURE
2. Raw full
ms
showing
full ms
patternwhich
of parent
compound
FIGURE
1. Workflow
tree
inspectrum
Compound
Discoverer
software
includes
14C labeling.
fromTracer
Pattern
node to create a trace for 14C compounds, Expected Finder node to
detect targeted transformation compounds and Unknown Detector node to detect
A2
untargeted compounds.
Compd_B_RLM_NADPH_t1h_240k #1714 RT: 17.86 AV: 1 NL: 8.98E6
F: FTMS + p ESI Full ms [150.0000-1000.0000]
474.24908
100
90
A3
80
70
60
40
containing metabolites?
The Pattern
Tracer node using pattern #3 (consisting of A0, A2, A3 and A4) effectively
Pattern
Selection
removed matrix background and other interferences. Metabolites containing 14C are
Three different patterns as shown in Figure 3 were used to extract out pattern traces.
revealed in the pattern traces when overlaying traces from T0 hr and T1 hr time points.
The results from the Compound Discoverer Pattern Tracer node
indicates the more
These metabolites are not visible in the overlaid base peak chromatograms. (Figure 5)
specific the pattern is, the better it removes matrix background. (Figure 4)
Base Peak Chromatogram (BPC; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t1h_240k.raw (976)
Base Peak Chromatogram (BPC; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t0_240k.raw (975)
2.0
20.688
20
472.24686
471.48642
471.5
472.0
473.25024
472.71927
472.5
473.0
473.5
474.11301
474.37082
474.88409
474.0
m/z
474.5
1.5
476.25522
475.0
475.5
476.0
Intensity [counts] (10^6)
10
0
476.5
17.238
1.0
16.933
13.534 13.717
13.580 13.656
Pattern trace using pattern#2: A0, A2 and A3
11.254
0.5
14.551
14.607 14.877
15.412
16.098
15.755
16.852
Pattern trace (Pattern; [custom pattern]; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t1h_240k.raw (976)
Intensity [counts] (10^6)
M3
Pattern trace using pattern#3: A0, A2, A3 and A4
M1
M2
1.5
1.0
16.626
M6
16.585
18.900
M7M8
19.454 19.962
15.714
12.116
14.327
14.326
19.982 20.175
20.103
19.875
16.117
12.074
18.571
18.491
18.800
18.351
18.431
19.020
14.714 14.917
14.768
18.391
19.353 19.440
19.273 19.360
18.980
19.313
19.975
19.922 20.203
20.163
20.405
19.741
19.734
19.233
0.0
10
A2
19.741
16.098
0.5
FIGURE 2. Raw full ms spectrum showing full
pattern
of parent
compound
2. ms
Pattern
consisting
A0, A
2 and A3
from 14C labeling.
19.501
19.360
19.320
18.311 18.371
18.251 18.540 18.720
M4 M5
Pattern trace (Pattern; [custom pattern]; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t0_240k.raw (975)
2.0
1. Pattern consisting A0 and A2 only
17.071 17.253
16.321 16.585
16.750 17.111
16.524
16.158 16.422
FIGURE 3. Isotope Ratio Editor in Pattern Trace node showing input of
experimental custom pattern.
Three different patterns were evaluated
12
14
16
18
20
RT [min]
Where are my 14C containing metabolites?
The
Pattern
Tracer node using pattern #3 (consisting of A0, A2, A3 and A4) effectively
What
are they?
removed matrix background and other interferences. Metabolites containing 14C are
Finding the
identities
thesewhen
metabolites
fromtraces
the pattern
was achieved easily
revealed
in the
patternoftraces
overlaying
from Ttrace
0 hr and T1 hr time points.
within metabolites
Compound Discoverer
software.
The workflow
used chromatograms.
to process the data
included
These
are not visible
in the overlaid
base peak
(Figure
5)
Expected Finder node which looks for modification compounds and Unknown Detector
node which detects compounds based on untargeted component detection. By manually
FIGURE
5. The
top plot peaks
showsonoverlaid
base
peakCompound
chromatograms
fromsoftware
T0hr and
integrating
the selected
the pattern
trace,
Discoverer
links
T1hr;
bottom
showsFinder
overlaid
traces
fromtoT0hr
and T1hr.integrated
peaksthe
detected
byplot
Expected
andpattern
Unknown
Detector
the manually
pattern trace peaks (Figure 6). m/z, compound explanations, isotope pattern fit score,
fragmentation ion match score, and spectral tree information became readily available to
help make the correct assignment of these compounds.
From Very High Resolution Mass Spectrometry
Compd_B_RLM_NADPH_t1h_240k #1714 RT: 17.86 AV: 1 NL: 8.98E6
F: FTMS + p ESI Full ms [150.0000-1000.0000]
100
474.24908
90
3. Pattern consisting
A0, A2, A3 and A4
A
80
3
70
60
A4
A0
50
men, Germany 3. Amgen Inc, South San Francisco, CA, USA
Base Peak Chromatogram (BPC; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t1h_240k.raw (976)
475.25128
30
471.5
472.0
472.71927
472.5
473.25024
473.0
473.5
Results
474.37082
474.88409
474.11301
474.0
m/z
474.5
475.0
475.5
476.0
20.688
1.5
476.25522
Intensity [counts] (10^6)
471.48642
472.24686
476.5
17.238
1.0
13.534 13.717
13.580 13.656
11.254
14.551
14.607 14.877
16.098
15.412
15.755
16.933
16.852 17.071 17.253
16.321 16.585
16.750 17.111
16.524
19.360
19.320
18.311 18.371
18.251 18.540 18.720
19.501
19.741
19.982 20.175
20.103
0.5
Pattern3.Selection
FIGURE
Isotope Ratio Editor in Pattern Trace node showing input of
experimental
custom
pattern.
Three different
patterns
as shown in Figure 3 were used to extract out pattern traces.
Three different patterns were evaluated
The results from the Compound Discoverer Pattern Tracer node indicates the more
1. Pattern
consisting
A0 and4)A2 only
specific the pattern is, the better it removes matrix
background.
(Figure
FIGURE 4. Pattern traces from different custom patterns
FIGURE 6. Manual peaks from pattern trace are linked with peaks detected by
Expected Finder and Unknown Detector nodes.
M4 M5
16.158 16.422
Pattern trace (Pattern; [custom pattern]; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t1h_240k.raw (976)
Pattern trace (Pattern; [custom pattern]; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t0_240k.raw (975)
2.0
Intensity [counts] (10^6)
10
Base Peak Chromatogram (BPC; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t0_240k.raw (975)
2.0
20
M3
M1
M2
1.5
1.0
16.626
19.875
M6
16.585
16.117
18.900
15.714
12.116
14.327
12.074
14.326
18.571
18.491
18.800
18.351
18.431
19.020
14.714 14.917
14.768
18.391
19.454 19.962
19.353 19.440
19.273 19.360
18.980
19.313
19.975
19.922 20.203
20.163
20.405
19.741
19.734
19.233
0.0
Pattern trace using pattern#1: A0 and A2 only
M7M8
16.098
0.5
10
12
14
16
18
20
RT [min]
2. Pattern consisting A0, A2 and A3
What are they?
Pattern trace using pattern#2: A0, A2 and A3
3. Pattern consisting A0, A2, A3 and A4
Pattern trace using pattern#3: A0, A2, A3 and A4
Finding the identities of these metabolites from the pattern trace was achieved easily
within Compound Discoverer software. The workflow used to process the data included
Expected Finder node which looks for modification compounds and Unknown Detector
node which detects compounds based on untargeted component detection. By manually
integrating the selected peaks on the pattern trace, Compound Discoverer software links
peaks detected by Expected Finder and Unknown Detector to the manually integrated
pattern trace peaks (Figure 6). m/z, compound explanations, isotope pattern fit score,
fragmentation ion match score, and spectral tree information became readily available to
help make the correct assignment of these compounds.
Fine isotopic structure confirmation for very high resolution data
Where are my 14C containing metabolites?
The Pattern Tracer node using pattern #3 (consisting of A0, A2, A3 and A4) effectively
removed matrix background and other interferences. Metabolites containing 14C are
revealed in the pattern traces when overlaying traces from T0 hr and T1 hr time points.
These metabolites are not visible in the overlaid base peak chromatograms. (Figure 5)
FIGURE 5. The top plot shows overlaid base peak chromatograms from T0hr and
T1hr; the bottom plot shows overlaid pattern traces from T0hr and T1hr.
Base Peak Chromatogram (BPC; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t1h_240k.raw (976)
The elemental composition proposals from Expected Finder for these putative 14C
containing compounds were confirmed by isotopic pattern fit calculation which is part of
the Expected Finder node. Since the parent compound contains 7 nitrogen atoms, and
these metabolites also contain the same number of nitrogen, the fine isotopic peaks
from 15N and 13C isotopes gave greater confidence in the final metabolite assignment
(Figure 7).
FIGURE 7. At 240K resolution, the 15N and 13C isotopes are resolved and visualized
within Compound Discoverer software.
#1588, RT=16.626 min, FTMS (+)
labeledcomdB + (Oxidation) C25 [14]C H29 N7 O3, MW: 489.23643, Area: 108879
Base Peak Chromatogram (BPC; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t0_240k.raw (975)
700
2.0
20.688
11.254
13.534 13.717
13.580 13.656
14.551
14.607 14.877
16.098
15.412
15.755
16.933
16.852 17.071 17.253
16.321 16.585
16.750 17.111
16.524
18.311 18.371
18.251 18.540 18.720
19.360
19.320
19.501
19.741
19.982 20.175
20.103
0.5
M4 M5
16.158 16.422
Pattern trace (Pattern; [custom pattern]; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t1h_240k.raw (976)
Pattern trace (Pattern; [custom pattern]; MS Order: MS1; Polarity: Positive;)
File: Compd_B_RLM_NADPH_t0_240k.raw (975)
2.0
16.626
19.875
Intensity [counts] (10^3)
Intensity [counts] (10^6)
17.238
1.0
490.24380
[M+H]+1
600
1.5
500
400
300
200
From Very High Resolution Mass 100
Spectrometry
2 Radio-Labeled Compound Detection Using
488.22787
M3Isotopic StructuresM6
489.05560
Intensity [counts] (10^6)
d
40
0
1.5
1.0
F
E
Pattern trace using pattern#1: A0 and A2 only
475.25128
30
14C
FIGURE
The top
plot shows
overlaidcustom
base peak
chromatograms from T0hr and
FIGURE
4. 5.
Pattern
traces
from different
patterns
T1hr; the bottom plot shows overlaid pattern traces from T0hr and T1hr.
A4
A0
50
Results
Where are my
M1
M2
M7M8
16.585
16.117
16.098
18.900
19.454 19.962
19.975
488.24069
491.24716
489.23700
489.24402
0
489
490.05490
491.24146
490
491
492.22281
492.25510
492.24420
492.25040
492
F
T
c
t
t
f
(
F
w
FIGURE 6. Manual peaks from pattern trace are linked with peaks detected by
FIGURE 6. Manual peaks from pattern trace are linked with peaks detected by
Expected Finder and Unknown Detector nodes.
Expected Finder and Unknown Detector nodes.
14C containing metabolites identified by Compound Discoverer software
14C containing metabolites identified by Compound Discoverer software
The following 14C14containing metabolites were detected by Compound Discoverer software
The following C containing metabolites were detected by Compound Discoverer software
using a single processing workflow employing Pattern Tracer, Expected Finder and Unknown
using a single processing workflow employing Pattern Tracer, Expected Finder and Unknown
Detector nodes (Table 1). Trace level metabolites were identified by this approach. The
Detector nodes (Table 1). Trace level metabolites were identified by this approach. The
smallest metabolite identified had a relative to parent area percent of 0.33%.
smallest metabolite identified had a relative to parent area percent of 0.33%.
In this study, all the 14C14containing peaks from pattern trace were explained by the Expected
In this study, all the C containing peaks from pattern trace were explained by the Expected
Finder node. The explanations from Expected Finder provided elemental composition,
Finder node. The explanations from Expected Finder provided elemental composition,
transformation, formula change, mass accuracy, retention time, isotopic pattern score and
transformation, formula change, mass accuracy, retention time, isotopic pattern score and
FISh coverage score. These information helped quicker and more confident metabolite
FISh coverage score. These information helped quicker and more confident metabolite
identification.
identification.
TABLE 1. 14C14containing metabolites including trace level metabolites identified by
TABLE 1. C containing metabolites including trace level metabolites identified by
Compound Discoverer software
Compound Discoverer software
Formula
Formula
P
Fine isotopic structure confirmation for very high resolution data
Fine isotopic structure confirmation for very high resolution data
The elemental composition proposals from Expected Finder for these putative 14C14
The elemental composition proposals from Expected Finder for these putative C
containing compounds were confirmed by isotopic pattern fit calculation which is part of
containing compounds were confirmed by isotopic pattern fit calculation which is part of
the Expected Finder node. Since the parent compound contains 7 nitrogen atoms, and
the Expected Finder node. Since the parent compound contains 7 nitrogen atoms, and
these metabolites also contain the same number of nitrogen, the fine isotopic peaks
these metabolites also contain the same number of nitrogen, the fine isotopic peaks
from 15N15and 13C13isotopes gave greater confidence in the final metabolite assignment
from N and C isotopes gave greater confidence in the final metabolite assignment
(Figure 7).
(Figure 7).
240K resolution, the 15N and 13C isotopes are resolved and visualized
At 240K resolution, the 15N and 13C isotopes are resolved and visualized
FIGURE 7. At
FIGURE 7.
within Compound Discoverer software.
within Compound Discoverer software.
Intensity [counts] (10^3)
Intensity [counts] (10^3)
600
500
400
300
200
100
0
700
490.24380
490.24380
[M+H]+1
[M+H]+1
600
M2
M3
M4
M5
M6
M7
M8
#1588, RT=16.626 min, FTMS (+)
#1588, RT=16.626
min, FTMS
(+) H29 N7 O3, MW: 489.23643, Area: 108879
labeledcomdB
+ (Oxidation)
C25 [14]C
labeledcomdB + (Oxidation) C25 [14]C H29 N7 O3, MW: 489.23643, Area: 108879
700
M1
C2514CH29N7O2
C2514CH29N7O2
473.24152
473.24152
M7
C2514CH29N7O3
C2514CH29N7O3
C2514CH29N7O3
C2514CH29N7O3
C2514CH31N7O3
C2514CH31N7O3
C2514CH29N7O3
C2514CH29N7O3
C2514CH29N7O3
C2514CH29N7O3
C2514CH29N7O3
C2514CH29N7O3
C2514CH29N7O4
C2514CH29N7O4
489.23643
489.23643
489.23643
489.23643
491.25208
491.25208
489.23643
489.23643
489.23643
489.23643
489.23643
489.23643
505.23134
505.23134
M8
C2614CH29N7O3
C2614CH29N7O3
501.23643
501.23643
P
M1
M2
M3
M4
M5
M6

400
300
489.23700
489.23700
489.05560
489.24402
490.05490
489.24402
490.05490
488.24069
0
489
490
489
490
m/z

491.24716
491.24716
200
488.22787
491.24146
491.24146
491
491
492.24420
492.22281
492.24420
492.22281492.25040
492.25510
492.25040
492.25510
492
492
Intensity [counts] (10^3)
Intensity [counts] (10^3)
600
500
400
300
200
100
0
700
600
500
400
300
200
100

m/z

#1588, RT=16.626 min, FTMS (+)
labeledcomdB
+ (Oxidation)
[14]C
#1588, RT=16.626
min,C25
FTMS
(+) H29 N7 O3, MW: 489.23643, Area: 108879
labeledcomdB + (Oxidation) C25 [14]C H29 N7 O3, MW: 489.23643, Area: 108879
700
--
--
Oxidation
Oxidation
Oxidation
Oxidation
Hydration
Hydration
Oxidation
Oxidation
Oxidation
Oxidation
Oxidation
Oxidation
Oxidation +
Oxidation
Oxidation +
Oxidation
Desaturation,
Desaturation,
Oxidation
+
Oxidation +
Methylation
Methylation
--
17.88
17.88
-0.10
1988333
-0.10
1988333
+(O)
+(O)
+(O)
+(O)
+(H2O)
+(H2O)
+(O)
+(O)
+(O)
+(O)
+(O)
+(O)
+(O2)
+(O2)
11.64
11.64
12.06
12.06
15.70
15.70
16.59
16.59
17.07
17.07
18.88
18.88
20.17
20.17
-0.26
6684
-0.26
6684
-0.20
15769
-0.20
15769
-0.21
27945
-0.21
27945
-0.14
108879
-0.14
108879
-0.33
39559
-0.33
39559
-0.26
55672
-0.26
55672
-0.41
42093
-0.41
42093
+(CO)
+(CO)
20.40
20.40
-0.26
-0.26
--
26039
26039
Conclusion
Conclusion
500
488.22787 489.05560
100488.24069
Monoisotopic Transformation Composition RT(min)
Mass
Area
Monoisotopic
Transformation Change
Composition RT(min)Accuracy
Mass
Area
mass
mass
Change
Accuracy
(ppm)
(ppm)
13C
13C

15N
15N

491.24716
491.24716
491.24146
Compound labeling combined with very high resolution LC/HRAM mass
 Compound labeling combined with very high resolution LC/HRAM mass
spectrometry is an effective way for confident compound detection and profiling
spectrometry is an effective way for confident compound detection and profiling
from complex biological samples
from complex biological samples
Compound Discoverer software provides a suite of advanced algorithms (nodes)
 Compound Discoverer software provides a suite of advanced algorithms (nodes)
which enable flexible yet powerful data processing that was previously not
which enable flexible yet powerful data processing that was previously not
possible.
possible.
The Pattern Tracer node is able to effectively reduce background and extract out
 The Pattern Tracer node is able to effectively reduce background and extract out
labeled compounds based on experimental custom pattern. When it is combined
labeled compounds based on experimental custom pattern. When it is combined
in a single workflow with peak detection mechanisms, compound identification
in a single workflow with peak detection mechanisms, compound identification
and profiling can be achieved without use of a radio detector.
and profiling can be achieved without use of a radio detector.
The pattern recognition algorithm in Compound Discoverer software is capable
 The pattern recognition algorithm in Compound Discoverer software is capable
of utilizing very high resolution data and fine isotopic structures, which gives user
of utilizing very high resolution data and fine isotopic structures, which gives user
greater confidence in results and helps get the answers quicker.
greater confidence in results and helps get the answers quicker.
The approach described here can be applied to any labeling studies.
 The approach described here can be applied to any labeling studies.
Future considerations include further improvement to the pattern search
 Future considerations include further improvement to the pattern search
algorithm and developing a mechanism to detect compounds based on custom
algorithm and developing a mechanism to detect compounds based on custom
pattern.
pattern.
491.24146
0
491.21
491.22
491.23
491.24
491.25
491.26
491.27
491.28
491.29
491.21
491.22
491.23
491.24 m/z491.25
491.26
491.27
491.28
491.29
o
m/z
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