Agilent MassHunter VistaFlux Software

TRACK METABOLIC FLUX

Metabolic flux analysis
Using different experimental conditions, flux analysis estimates the rates of metabolic change and canhighlight affected pathways. Changes in flux are the result of either differences in the amount of enzyme
present or activity of the enzyme. In addition, flux analysis can expose unexpected shunts between
metabolites in well-studied pathways.

Qualitative flux analysis
While there are many approaches for quantitative metabolic flux analysis, they all require hours of expert
work (through many experiments and iterations) to refine the model and return a result. Qualitative flux
analysis, on the other hand, allows you to quickly obtain information about selected pathways and fluxes
without the challenging task of constructing quantitative models.
Qualitative flux analysis traces isotopologues and targets specific pathways of interest by acquiring data on
a mass spectrometer. By mining the data for pathway-targeted metabolites and incorporating stable isotope
labeling, you’ll be able to follow metabolic fluxes—then use graphical tools to visualize the results.

However, the state of the art in qualitative flux analysis still requires
practitioners to deal with several challenges:
ACQUIRE
generating LC/MS data with highmass-accuracy and isotopic fidelity
EXTRACT
mining target metabolites ions,including stable isotope labeledions (isotopologues)
VISUALIZE
viewing and sharing results on
pathways to accelerate biologicalinterpretation2

INTRODUCING MASSHUNTER VISTAFLUX“Qualitative 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. Fluxes are not readily predictablefrom proteome and metabolome data. Assessing fluxes by stable isotopic
tracers is a powerful approach to study the metabolic network in motion.”
NICOLA ZAMBONI, PhD, INSTITUTE FOR MOLECULAR SYSTEMS BIOLOGY, ETH ZURICH

MassHunter VistaFlux software is designed to meet the challenges of qualitative flux analysis.
Using both static and animated pathway visualizations, it enables you to easily interpret and explain
the results of the experiments you conduct using Agilent time-of-flight and quadrupole time-of-flight
LC/MS systems.
VistaFlux easily finds targeted metabolites containing stable isotope labeled data, and displays
results using advanced pathway visualization software. In addition, it exports figures forpresentations and generates reports, making the complete workflow easy and fast.

VistaFlux workflow
Create Target
ResultAcquire
Data

Extract
Isotopologues

Visualize onPathways

Results3

DATA MINING DESIGNED FOR QUALITATIVEFLUX ANALYSIS

MassHunter Profinder software is a batch-processing module of VistaFlux that provides fast
reviews and visualization of metabolite isotopologues. The Profinder module provides grouping of
replicate data files, binning and alignment, multiple chromatogram overlay and color by sample
group, manual reintegration of peaks, and file export for differential analysis in Agilent MassHunter
Mass Profiler Professional. What’s more, Profinder eliminates tedious manipulation of data andmanual calculations in spreadsheets.

Batch isotopologue extraction

Extracted spectrumof unlabeled citrate

Profinder extraction
results summary

Batch isotopologue extraction in Profinder is designed specifically for qualitative flux dataacquired on Agilent high-resolution, accurate-mass, time-of-flight LC/MS systems. Batch
isotopologue extraction isolates ions based on metabolite chemical formulas. Based on thetarget list, the Profinder algorithm mines for stable isotope-labeled ions (isotopologues) andsupports 13C, 15N, or 2H stable isotope labeling.

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Extracted spectrumof 13C labeled citrate

PATHWAY VISUALIZATION FOR FLUX ANALYSIS

Results from Profinder flow into Omix Premium, another module of VistaFlux, for effortlessvisualization of metabolic fluxes. Omix Premium enables you to:Effortless Visualization• Interactively visualize flux data on
pathway maps.
• Animate metabolic flux through pathways
for time-course studies.
• Easily leverage publicly available
pathway databases.
• Customize and connect
metabolic pathways.
• Tailor the visualization to display different
information on pathway diagrams.
• Export figures and animations forpublication and presentations.

MassHunter Profinder

Omix Premium“Flux analysis and visualization give researchers theopportunity to mine protein activity. This is whatmetabolomics is all about. Qualitative flux is a hugeleap forward to discover protein activity.”
ANDERS NORDSTRÖM, PhD, DEPARTMENT OF
MOLECULAR BIOLOGY, UMEÅ UNIVERSITY

5COMPREHENSIVE VISUALIZATIONS FORFLUX EXPERIMENTS
There are many types of metabolic flux experiments, each requiring different information to interpret theresults. VistaFlux allows you to tailor the information displayed on the metabolic pathway to effectively
communicate your results. With VistaFlux, you can include as little or as much detail as you need.

VistaFlux display options
2-oxoglutarate

OO-0 hr O
O- hr
0.5O-O0 hr2-oxoglutarate2-oxoglutarateO-* 0.5 hr * *1 hr*
1 hrOO**O
3 hrO*
3 hr***e 2-oxoglutarate8 hr2-oxoglutarate8 hr**

0 hr
0.5 hr

*
*
3 hr
*
8 hr
*
1 hr

**
*
*

2-oxoglutarate
0 hr

* *
*
2-oxoglutarate2-oxoglutarate
1 hr * **3 hr * **8 hr * **0.5 hr+0 +1 +2 +3 +4 +5+0 +1 +2 +3 +4 +5+0 +1 +2 +3 +4 +5+0 +1 +2 +3 +4 +5

Showing chemical structure
on pathway

Quilt plot view of theisotopologue abundances

Labeling Inc
Labeling Incorporation
Labeling Incorporation
0%30%
0%30% 60%100%
0%30% 60%
100%
0 hr
0 hr
0 hr
0.5 hr
0.5 hr
0.5 hr2-oxoglutarate
1 hr
1 hr 2-oxoglutarate
1 hr
3 hr
3 hr
3 hr
8 hr
8 hr
8 hr
Labeling
Labeling Incorporation
Total Abundance Labeling Incorporation Total Ab
Total Abundance

Filling metabolite nodes toindicate fold-change (left),label incorporation (middle) andfractional labeling (right)

Plotting isotopologue
incorporation as a histogram“In our short time using Agilent’s VistaFlux software, we havesuccessfully processed many qualitative flux projects employing
13
C or 15N stable isotope tracers. We like the simplicity, speed, andperformance of the software. It has quickly become an important
tool in my laboratory for analyzing Agilent’s data sets.”
GARY PATTI, PhD, DEPARTMENT OF CHEMISTRY,WASHINGTON UNIVERSITY, USA

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VISUALIZATIONS AS VIBRANT AS YOUR DATAMetabolism is in constant flux, and time-course studies attempt to capture this fluidity. In addition tostatic images, the Omix Premium module provides dynamic visualizations that make time-course resultseasier to interpret. You can share these animations as movie files or embed them into presentations.

Dynamic visualizations
Omix Premium supports easy visualization oftime-course studies with step, play or record
modes. The figure above shows the flux at
0.5, 1, 3, and 8-hour time points (from upperleft to bottom right) for 2-oxoglutarate and2-hydroxyglutarate.

0.5 hour

1 hour3 hour8 hourVistaFlux transforms your metabolomics research by reducing the difficulty of stable isotope labeling.
The software converts a time-consuming, labor-intensive process into a powerful qualitative flux
workflow, including metabolite isotopologue extraction and pathway visualization.
In short, VistaFlux lowers the barriers to doing qualitative metabolic flux experiments
to increase your biological understanding.

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Learn more
www.agilent.com/chem/vistaflux
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For Research Use Only. Not for use in diagnostic procedures.
RA45201.6430555555
This information is subject to change without notice.
© Agilent Technologies, Inc., 2016, 2023
Published in the USA, October 20, 2023
5991-7341EN