extend your metabolomics insight! · 2016-10-10 · 3. batch targeted feature extraction • also...

Extend Your Metabolomics Insight!

Introducing MassHunter VistaFlux

Dr. Ben Owen

MS Product Specialist,

Upper Midwest

Agilent Technologies

Agilent Solutions for Metabolomics

– A broad family of products (LC/MS, GC/MS, CE/MS and SFC/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

For Research Use Only. Not for use in diagnostic procedures. May 27, 2016

MassHunter Vistaflux

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Agilent Instrumentation For Metabolomics

QQQ

6400 Series

Q-TOF

6500 series

TOF

6200 series

1290

Infinity II

UHPLC

Hi-DEF Q-TOF

6500 series

7200B

GC/Q-TOF

7000C

GC/QQQ

5977A

GC/MS

7100 CE1260 SFC



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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



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Agilent – THE Leader in Metabolomics!

Fiehn EI Library

METLIN MS/MS Library

Mass Profiler Professional with Pathway Architect

ProfinderVista Flux

Agilent Innovations for

Metabolomics



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MassHunter ProfinderHigh-throughput compound finding

MassHunter Profinder is a

productivity tool for processing

multiple samples in profiling

analyses

Fast Compound Finding

• Untargeted using MFE

• Targeted using Find by Formula

Visualize, review, and edit results

by compound across many

samples

Higher quality results based on

cross-sample processing

Aligned

EICs of

six

replicate

injections

May 27, 2016


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MassHunter Profinder - Main ViewOnly Four Linked Windows

Compound Group Table

Compound Details Table

EIC’s Spectra

May 27, 2016


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1. Batch Molecular Feature Extraction

• Recursive MFE reduces false positives

2. Batch Recursive Feature Extraction

• Greater “missing feature” recovery

• Allows manual editing of compounds

3. Batch Targeted Feature Extraction

• Also allows manual editing

• Data source

Profinder’s Three Feature Extraction Algorithms

MFE rMFE

Find by Ion

Find by Formula

MFE rMFE

May 27, 2016


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Molecular Feature Extraction

• Batch Molecular Feature Extraction begins with MFE on

each data file

• Features are extracted as in MassHunter Qualitative

Analysis, one file at a time

• Batch MFE then aligns across all sample files

raw groupedfiltered features aligned

MFE

May 27, 2016


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Why Batch MFE?

• MFE can sometimes assign different adduct species for each data file

• As an example, in one file the 537 species was identified as M+H, but in the other there was sufficient evidence to declare it M+Na

• Batch MFE will correct the assignment to M+Na, given the consensus assignment across all sample files

• This technique reduces false positives by pulling ONLY signal out of noise

537.3950 as M+H

537.3950 as M+Na

rMFE

May 27, 2016


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• Find by Ion reduces false negatives




• Data source can be .CEF files, a .CSV file


MFE rMFE

Find by Ion

Find by Formula

MFE rMFE

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• Find by Ion reduces false negatives




• Formula source can be .CEF files, a .CSV file or a PCD/L


MFE rMFE

Find by Ion

Find by Formula

MFE rMFE

May 27, 2016


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MassHunter Profinder:Manual Editing of Data

Designed for Easy Manual Editing of Data Results

Originally Selected Compounds

Manually Selected Compounds

Tables Automatically Update

May 27, 2016


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MassHunter Profinder Mass Profiler ProfessionalAgilent LC/MS & GC/MS

process discoverdetect

MassHunter Profinder B.08.00High Performance Feature Extraction for LC/MS & GC/MS



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Untargeted Feature Extraction for GC/Q-TOF & GC/MSDGC MFE + Recursive Feature Extraction

• Using GC MFE as a first-pass feature finding step allows us to take advantage of the already-existingFind by Ion that benefits LC-QTOF today

• This provides a single, cohesive workflow for both LC/MS and GC/MS


MPPProfinder

+GC MFE

+Find by Ion



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Targeted Feature Extraction for GC/Q-TOFHigh Performance All Ions Feature Extraction

• Targeted feature finding

for GC-QTOF is

performed using the EI

spectral All Ions

algorithm

• Using a PCDL EI

spectral library, All Ions

performs targeted

fragment confirmation

and feature extraction


MPPProfinder + PCDL



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Profinder B.08.00 Performance Time-LapseMultithreading Demo – 195 files, ~4:31 min/file avg.

• 14 hours and 44 minutes on 195 files

translates to an average of 4 minutes, 31 sec per file – faster than most

chromatography!

• This multithreading optimization is applied to all algorithms, both

untargeted and targeted, for LC/MS and GC/MS workflows



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Accelerate Your Research With VistaFlux! Agilent’s Qualitative Flux Analysis Workflow

Extend your

metabolomics insight …

Track the flow of metabolites

through a pathway!

Succinate

Citrate

Isocitrate

α-Ketoglutarate

Succinyl CoAFumarate

Malate

Oxaloacetate

VistaFlux



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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

??



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VistaFlux Stable Isotope TracingIsotopologue Tracking

Use of stable isotope labels (13C, 15N, and 2H)

Monitor stable isotope incorporation

Fumarate C4H4O4

Isotopologues

M+0: 13C0C4H4O4

M+1: 13C1C3H4O4

M+2: 13C2C2H4O4

M+3: 13C3C1H4O4

M+4: 13C4C0H4O4

CHO

C

C

C

OH

O

O

CHO

C

C

C

OH

O

O

CHO

C

C

C

OH

O

O

CHO

C

C

C

OH

O

O

Isotopomers of M+1

0 hr

8 hr



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Manual Qualitative Flux Analysis – a Tedious Process!Current Flux Data Analysis Workflow

Create target metabolite list

Determine m/zfor 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.



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Accelerate Your Research With VistaFlux! Agilent’s Qualitative Flux Analysis Workflow

• Pathways to PCDL

• PCDL Manager

Create Target Metabolite List

• MassHunter Acquisition

• LC/TOF or LC/Q-TOF

Acquire Data

• Profinder

Extract Features

• OmixPremium

Visualize on Pathways

VistaFlux Workflow



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Target List Creation in VistaFlux

Target List

Creation

Specify Interesting Pathways

Generate Metabolite

List

Determine Tracer



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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? 6545 LC/Q-TOF

(best metabolite

performance)

6230 LC/TOF

(best value)

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



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Data Analysis in VistaFluxProfinder Batch Isotopologue Extraction

Extract Features

Targeted Data Mining for

Metabolites

Score by Isotopologues and Retention

Time



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Data Analysis in VistaFluxSophisticated 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



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Isotopologue Extraction ExampleFirst Stage Extraction – Wider m/z and RT Windows

1

2

(3)

4

M-1

M+0

M+1

M+2

M+3

M+4

M+0

M+1

M+4

M+5

M+6

M+7



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Challenging Isotopologue Extraction ExampleSecond Pass Extraction – Narrower m/z and RT Windows

1

4

3

(2)

Sum

M+0

M+1

M+4

M+5

M+6

Qualified Ions After 2nd Pass

M+0

M+1

M+0

M+1

M+4

M+5

M+6

M+6• 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



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Visualize Profinder Results on PathwaysOmix Premium Visualization

Visualize on Pathways

Display Isotopologue

Metabolite Data

Static Pictures

Play Video of Time

points/networks



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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



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Qualitative Flux Analysis ExperimentTargeted Isotopologue Extraction Results

For Research Use Only. Not for use in diagnostic procedures.


MS Spectra Isotopologue Results by Time Point

May 27, 2016


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Qualitative Flux Analysis ExperimentQuick Summary of Isotopologue Results on Network



• 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

Abundance Stable Isotope

Incorporation

Both Abundance and

Incorporation

May 27, 2016


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Qualitative Flux Analysis ExperimentOptional 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-13C-Gln

• Demonstrates glutamine is the major substrate used to produce 2-HG in IDH2-mutant CS-1 cells.



May 27, 2016


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Qualitative Flux Analysis ExperimentOptional 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



May 27, 2016


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Qualitative Flux Analysis ExperimentComprehensive View of Isotopologue Results on Network



May 27, 2016


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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



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extend your metabolomics insight! · 2016-10-10 · 3. batch targeted feature extraction • also...

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