Extend Your Metabolomics Insight!

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Mervyn McCormick
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1 Extend Your Metabolomics Insight! Introducing MassHunter VistaFlux April 2016

2 Agilent THE Leader in Metabolomics! Fiehn EI Library METLIN MS/MS Library Mass Profiler Professional with Pathway Architect Profinder Vista Flux Agilent Innovations for Metabolomics 2

3 Accelerate Your Research With VistaFlux! Agilent s Qualitative Flux Analysis Workflow Oxaloacetate Citrate Isocitrate Extend your metabolomics insight VistaFlux Track the flow of metabolites through a pathway! Malate α-ketoglutarate Fumarate Succinyl CoA Succinate 3

4 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?? 4

VistaFlux Stable Isotope Tracing Isotopologue Tracking Use of stable isotope labels ( 13 C, 15 N, and 2 H) Monitor stable isotope incorporation Isotopologues 0 hr M+0: 13 C 0 C 4 H 4 O 4 M+1: 13 C 1

5 VistaFlux Stable Isotope Tracing Isotopologue Tracking Use of stable isotope labels ( 13 C, 15 N, and 2 H) Monitor stable isotope incorporation Isotopologues 0 hr M+0: 13 C 0 C 4 H 4 O 4 M+1: 13 C 1 C 3 H 4 O 4 M+2: 13 C 2 C 2 H 4 O 8 hr 4 M+3: 13 C 3 C 1 H 4 O 4 M+4: 13 C 4 C 0 H 4 O 4 Isotopomers of M+1 O HO C C C C OH O O HO C C C C OH O O HO C C C C OH O O Fumarate C 4 H 4 O 4 HO C C C C OH O 5

6 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. 6

7 Accelerate Your Research With VistaFlux! Agilent s Qualitative Flux Analysis Workflow Pathways to PCDL PCDL Manager Create Target Metabolite List Acquire Data MassHunter Acquisition LC/TOF or LC/Q-TOF Profinder Extract Features Visualize on Pathways Omix Premium VistaFlux Workflow 7

8 Target List Creation in VistaFlux Target List Creation Specify Interesting Pathways Generate Metabolite List Determine Tracer 8

Acquire Data on Agilent LC/TOF or LC/Q-TOF Acquire Data Use Accurate Mass TOF/Q-TOF

complex samples Isotopic Fidelity Detect incorporation of isotopic label through metabolic

9 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 (best metabolite performance) 6230 LC/TOF (best value) 9

10 Data Analysis in VistaFlux Profinder Batch Isotopologue Extraction Extract Features Targeted Data Mining for Metabolites Score by Isotopologues and Retention Time 10

11 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 11

12 Isotopologue Extraction Example First Stage Extraction Wider m/z and RT Windows M-1 M+0 M+1 M+2 M+3 M+4 M+0 M+1 M+4 M+5 M+6 M (3) 12

Challenging Isotopologue Extraction Example Second Pass Extraction Narrower m/z and RT Windows Qualified Ions After 2 nd Pass Sum M+0 M+1 M+4 M+5 M+6 M+0 M+1 1 (2) M+0 M+1 M+4 M+5 M+6 3 M+6

13 Challenging Isotopologue Extraction Example Second Pass Extraction Narrower m/z and RT Windows Qualified Ions After 2 nd Pass Sum M+0 M+1 M+4 M+5 M+6 M+0 M+1 1 (2) M+0 M+1 M+4 M+5 M+6 3 M+6 4 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 13

14 Visualize Profinder Results on Pathways Omix Premium Visualization Visualize on Pathways Display Isotopologue Metabolite Data Static Pictures Play Video of Time points/networks 14

15 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 15

16 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 For Research Use Only. Not for use in diagnostic procedures. April 29,

Qualitative Flux Analysis Experiment Quick Summary of Isotopologue Results on Network

coloring gives quick summary on network For this experiment, confirms that 2-HG is

Justin Cross, Donald B. and Catherine C.

17 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 For Research Use Only. Not for use in diagnostic procedures. April 29,

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 13 C

18 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 13 C 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- 13 C- Gln 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 For Research Use Only. Not for use in diagnostic procedures. April 29,

19 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 For Research Use Only. Not for use in diagnostic procedures. April 29,

20 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 For Research Use Only. Not for use in diagnostic procedures. April 29,

21 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 21

22 Agilent Instrumentation For Metabolomics 7000C GC/QQQ 5977A GC/MS 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 22

for statistically different features Annotate and identify interesting features Search

23 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 23

24 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 24

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Agilent MassHunter Profinder: Solving the Challenge of Isotopologue Extraction for Qualitative Flux Analysis

Agilent MassHunter Profinder: Solving the Challenge of Isotopologue Extraction for Qualitative Flux Analysis Technical Overview Introduction Metabolomics studies measure the relative abundance of metabolites