Extend Your
Metabolomics Insight!
Introducing MassHunter
VistaFlux
April 2016
Agilent – THE Leader in Metabolomics!
Profinder
METLIN
MS/MS
Library
Fiehn EI
Library
Vista
Flux
Mass Profiler
Professional
with Pathway
Architect
Agilent Innovations for
Metabolomics
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
2
Accelerate Your Research With VistaFlux!
Agilent’s Qualitative Flux Analysis Workflow
Citrate
Oxaloacetate
Isocitrate
Extend your
metabolomics insight …
Track the flow of metabolites
through a pathway!
VistaFlux
α-Ketoglutarate
Malate
Fumarate
Succinyl CoA
Succinate
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
3
The Importance of Qualitative Flux Analysis
Extend Your Metabolomics Insight
“Flux analysis elevates
metabolic studies to a different
level. While metabolomics
reports on the molecular
composition of a cell, flux
analysis reveals in vivo pathway
activity.”
- Nicola Zamboni, PhD, Institute for
Molecular Systems Biology, ETH Zurich
?
?
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
4
VistaFlux Stable Isotope Tracing
Isotopologue Tracking
Use of stable isotope labels (13C, 15N, and 2H)
Isotopomers of M+1
Monitor stable isotope incorporation
O
HO
Isotopologues
0 hr
M+0: 13C0C4H4O4
M+1:
8 hr
M+2:
13C
13C
C
C
HO
1C3H4O4
O
C
C
C
HO
O
C
M+3: 13C3C1H4O4
C
M+4: 13C4C0H4O4
O
C
OH
C
O
C
C
OH
C
O
2C2H4O4
OH
C
O
HO
Fumarate C4H4O4
C
C
C
OH
O
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
5
Manual Qualitative Flux Analysis – a Tedious Process!
Current Flux Data Analysis Workflow
Create target
metabolite list
Determine m/z
for all possible
isotopologues
Extract
chromatogram
for all
isotopologues
for all targets
Integrate peaks
and transfer
abundance to
spreadsheet
Review results
Manually
visualize on
pathways using
Cytoscape,
Adobe
Illustrator etc.
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
6
Accelerate Your Research With VistaFlux!
Agilent’s Qualitative Flux Analysis Workflow
• Pathways to
PCDL
• PCDL
Manager
Create Target
Metabolite List
Visualize on
Pathways
Acquire Data
• MassHunter
Acquisition
• LC/TOF or
LC/Q-TOF
• Profinder
• Omix
Premium
Extract
Features
VistaFlux Workflow
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
7
Target List Creation in VistaFlux
Target List
Creation
Specify
Interesting
Pathways
Generate
Metabolite
List
Determine
Tracer
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
8
Acquire Data on Agilent LC/TOF or LC/Q-TOF
Acquire
Data
Use Accurate
Mass
TOF/Q-TOF
Separate
Isobaric
Metabolites
with RT
What measurement factors
are important in flux
analysis?
Mass Accuracy and High Resolution
Confidently track isotopologues of metabolites in
complex samples
Isotopic Fidelity
Detect incorporation of isotopic label through
metabolic pathways
Dynamic Range
Detect low levels of stable isotope incorporation
in the presence of other intense isotopologues
6545 LC/Q-TOF
6230 LC/TOF
(best metabolite
performance)
(best value)
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
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
9
Data Analysis in VistaFlux
Profinder Batch Isotopologue Extraction
Extract
Features
Targeted Data
Mining for
Metabolites
Score by
Isotopologues
and Retention
Time
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
10
Data Analysis in VistaFlux
Sophisticated Multi-stage Isotopologue Extraction Algorithm
For each target metabolite:
• Extracts chromatogram for each possible isotopologue using specified RT range and
wider m/z tolerance
• Integrates sum of EICs and evaluates to narrow RT range
• Extracts MS and disqualifies ions not meeting criteria (m/z match and elution profiles)
• Creates new, combined EIC from sum of qualified ions
• Scores remaining peaks based on number of qualified isotopologue ions, relative
abundance, and distance from target RT
• Determines raw isotopologue abundance
• Calculates corrected isotopologue abundance using natural isotope abundance and
purity of the isotope tracer
Benefits:
• Minimizes false positive results
• Provides more accurate isotopologue abundances, which yields more precise results
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
11
Isotopologue Extraction Example
First Stage Extraction – Wider m/z and RT Windows
M-1
M+0
M+1
M+0
M+1
M+4
M+5
M+2
M+3
M+4
M+6
M+7
1
4
2
(3)
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
12
Challenging Isotopologue Extraction Example
Second Pass Extraction – Narrower m/z and RT Windows
Qualified Ions After 2nd Pass
M+6
Sum
M+0
M+1
M+4
M+5
M+6
4
M+0
M+1
M+4
M+5
M+6
M+0
M+1
1
3
•
Peak 2 has no qualified ions
after narrow window extraction
•
M+6 fails elution profile test so
peak 4 is eliminated
•
Peak 3 wins – closest to
expected RT and has the most
isotopologue peaks
(2)
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
13
Visualize Profinder Results on Pathways
Omix Premium Visualization
Visualize on
Pathways
Display
Isotopologue
Metabolite Data
Static Pictures
Play Video of
Time
points/networks
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
14
Qualitative Flux Analysis Experiment:
Cancer Cell Line with Enzyme Mutation (IDH2)
Goal: Understand impact of
mutation on glutamate
metabolism
Tracer experiment with
U-13C-Glutamine (Gln)
Monitoring metabolites
labeled in green
Courtesy of Justin Cross, Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
15
Qualitative Flux Analysis Experiment
Targeted Isotopologue Extraction Results
MS Spectra
Isotopologue Results by Time Point
Courtesy of Justin Cross, Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
16
Qualitative Flux Analysis Experiment
Quick Summary of Isotopologue Results on Network
Abundance
Stable Isotope
Incorporation
Both Abundance and
Incorporation
• Optional node coloring gives quick summary on network
• For this experiment, confirms that 2-HG is produced by the
mutant IDH2 enzyme from glutamine via α-KG via the TCA
cycle
Courtesy of Justin Cross, Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
17
Qualitative Flux Analysis Experiment
Optional Detailed Views of Isotopologue Results on Network
•
Quilt plot displays all time points
and isotopologue results
•
Fractional labeling shows
weighted 13C incorporation
For this experiment:
•
Significant label incorporation
into M+5 isotopologue of 2-HG
•
Incorporation occurs after 3 and
8-hours of incubation with U-13CGln
•
Demonstrates glutamine is the
major substrate used to produce
2-HG in IDH2-mutant CS-1 cells.
Courtesy of Justin Cross, Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
18
Qualitative Flux Analysis Experiment
Optional Detailed Views of Isotopologue Results on Network
• Isotopologue results with error bars for standard deviation
• Shows cellular glutathione pool becomes labeled with first one
and then two Gln-derived 5 carbon building blocks.
• Node fill indicates this is a highly abundant metabolite, thus it
takes time to impact the total cellular pool
Courtesy of Justin Cross, Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
19
Qualitative Flux Analysis Experiment
Comprehensive View of Isotopologue Results on Network
Courtesy of Justin Cross, Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
20
Agilent Solutions for Metabolomics
– A broad family of products (LC/MS, GC/MS and CE/MS) to meet your needs
– Agilent TOF technology is most-suited for discovery metabolomics
• High mass accuracy and mass stability
• Wide dynamic range with good resolution and high isotope ratio fidelity
• Highest sensitivity for low level metabolite detection
– Profinder is the most advanced software for high quality feature finding
– METLIN is the largest commercially available MS/MS library for metabolites
– MSC enables identification of MS/MS spectra without library matches
– Software designed to analyze metabolomics data
– Pathway compound ID mapping using automated Agilent BridgeDB
– Ability to visualize metabolomics data in pathways
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
21
Agilent Instrumentation For Metabolomics
5977A
GC/MS
7000C
GC/QQQ
7100 CE
1260 SFC
Hi-DEF Q-TOF
6500 series
Q-TOF
6500 series
7200B
GC/Q-TOF
1290
Infinity II
UHPLC
QQQ
6400 Series
TOF
6200 series
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
22
Most Extensive Software Portfolio for Metabolomics
Data Analysis
Find features across many
complex data files
Align extraction results from
many files
Analyze extracted data for
statistically different features
Annotate and identify interesting
features
Search and map annotated
metabolites to find interesting
pathways
Visualize results on pathways
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
23
Summary
• The new Agilent VistaFlux workflow extends metabolomics
capability by adding qualitative flux analysis
• Researchers will get fast, reliable results that can easily be
shared with others through pathway diagrams and videos
• VistaFlux is the latest addition to Agilent’s comprehensive
metabolomics portfolio of hardware, software, consumables,
and support
“Profinder allows users fast data mining and isotopologue
identification. The ability to frame and visualize flux data in Omix
Premium facilitates interpretation across multidisciplinary teams.”
- Dr. Pietro Morlacchi, Research Scientist, MD Anderson Cancer Center
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
24
MassHunter VistaFlux
For Research Use Only. Not for use in diagnostic procedures.
April 29, 2016
25