simplifying the routine lc/ms analysis of central carbon ...€¦ · simplifying the routine lc/ms...

Simplifying the Routine LC/MS Analysis of Central Carbon Metabolites

Christine Miller

Omics Market Manager

June 8, 2016

Enabling Metabolomics Research

Agilent Technologies is the leading company with platforms and software that support metabolomics research

• Team of knowledgeable scientists dedicated to metabolomics

• Broad instrument portfolio (LC/MS, CE/MS, GC/MS, SFC/MS)

• Common MS raw data processing software

• Single statistics analysis software — use the same visualization andstatistical analysis tools for your metabolomics (and multi-omics!) study

• New MassHunter VistaFlux for qualitative flux analysis

• Seahorse technology for cellular metabolic analysis

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Metabolomics dMRM Database and MethodIntegrated with Agilent’s Metabolomics Workflows

Database covers >215 central carbon metabolites

Designed for 1290 Flex pump and 6460/6470 QQQ LC/MS Systems

Provides an optimized method and database with stable, robust chromatography

Agilent Omics Workflows

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QQQ DataAcquisition

&Analysis

QuantitationSeparate & Detect

StatisticsPathways

LC/QQQ Quant

Mass Profiler ProfessionalPathway Architect

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

Metabolomics by Mass SpectrometryData Acquisition & Analysis

Targeted Data Acquisition – QQQ• Known metabolites only• Higher sensitivity than profiling approach• Absolute quantitation • Typically develop methods for tens/hundreds of metabolites• Project results onto biological pathways for interpretation

Discovery (Untargeted) Data Acquisition – TOF / Q-TOF• Acquire data and detect all compounds; relative quantitation.• Targeted data mining: − Analyze data with a database of known compounds− Project results onto pathways

• Untargeted data mining: − Find all compounds − Track metabolites using mass, or mass spectra and retention time

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Benefits of QQQ Technology for Targeted Metabolomics

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• Detect low level metabolites• Reduce sample required for analysisHigh Sensitivity

• Minimize matrix interference• Confirm compound identificationHigh Selectivity

• Analyze more compounds in a single run• Support fast chromatography

Fast MRM Transitions

• Provide reliable quantitation for differential analysis across a broad range of concentrations

Broad Dynamic Range

Metabolomics dMRM Database and MethodLC/QQQ SystemAgilent 1290 Infinity II LC system: • Infinity II Flex pump• Multisampler with chiller• Multicolumn Thermostat with 2-

position/6-port ultra high pressure valve

Agilent 6460 or 6470 Triple Quadrupole LC/MS system

Includes LC/MS method, database, quantitation method, and user guide

March 14, 2016

US HUPO 2016

LC/MS Method: Reverse Phase Ion Pair Chromatography• Better separation for acidic compounds

• Better protection for sugar phosphates and nucleotides

• Excellent retention time reproducibility across large sample sets

However: • Pump must be dedicated to the QQQ as ion pair reagents

stick to parts and cannot be effectively cleaned

• MS source can be easily cleaned allowing positive mode work

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Chromatographic Separation of Acidic MetabolitesNegative Mode Ionization

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• Mobile phase: solvent A (97:3 water/methanol with 10 mM tributylamine + 15 mM acetic acid) and solvent B (97:3 acetonitrile/methanol with 10 mM tributylamine + 15 mM acetic acid).

• Columns: Zorbax RRHD Extend-C18 2.1 x 150 mm column and guard column

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Ion Pair Method Separates Biologically Relevant Isomers

IsoleucineLeucine

Glucose-6-phosphate

Glucose-1-phosphate

Demonstrated Robustness of Reverse Phase Ion Pair Chromatography

RT Reproducibility After 7 Months (> 4000 sample injections!)

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RT Deviation for 62 different samples over 3 days

RT for fresh column

RT

afte

r >40

00 s

ampl

es

Average RT across all samples

RT

acro

ss a

ll sa

mpl

es

Database: Curated MRM Transitions

The compound database with MRM transitions was developed together with the Rosebrock LabCompound and MRM transitions are extensively curated

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Curation verifies these entries for correctness:Database MRM transitions

• Compound common name• MRM transitions (precursor and product m/z)• Fragmentor voltage• Collision energy• Retention time and retention time window• Unit mass of neutral molecule• Molecular formula• CAS number of the native compound

• MRM transitions have been optimized by collaborators

• Agilent curates dMRM data provided by Rosebrock lab and creates Master database and documentation.

The curated database eliminates the need to develop MRM transitions and retention times for a large numbers of compounds


Easy Transfer From Database to Method in MassHunter Acquisition

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Simple Quantitative Batch Review and Export to MPP

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MassHunter Quant Batch Review

Export to MPP


Metabolomics is Uniquely Suited for Pre-built Methods: Compounds of Interest are Shared Across Organisms

The same metabolites and pathways are present across many organisms, allowing use of one method for many biological samples and questions.

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Data from Adam Rosebrock Lab, University of Toronto


Bacteria Yeast Tissues, cells, and biofluids

Customer Application: Antibiotic Mode of Action Discovery by Targeted Metabolomic Analysis

Objectives• Determine effects of drug perturbation on the metabolic network

• Generate metabolic fingerprints to characterize the mechanism of

action of novel biologically active molecules

Experimental approach• E. coli MG1655

• Synthetic minimal media (M9 glucose-ammonia)

• Dose-response design at a single time point

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Example Application of Metabolomics dMRM Method: Identifying Metabolic Effects of Small Molecule Treatment

Inhibition of metabolism causes predictable changes in metabolite levels

Metabolites upstream of the blockade accumulate, while downstream metabolites are depleted

Typically, multiple compounds upstream and downstream are affected, allowing identification of the inhibited pathway even in cases where not all metabolites can be measured

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XCompound 1 Compound 2 Compound 3 Compound 4 Compound 5

Targeted Metabolomics Analysis Reveals Dose-dependent Biochemical Responses to Drug Treatment

• E. coli cultures were treated in replicate across a dose-response series as shown

• Cultures were processed as detailed in the method quick-start guide.

• Data were extracted in MassHunter Quant and processed in Mass Profiler Professional

• Heat-map displaying relative changes in each of ~215 metabolites is shown here. Dynamic range is 64-fold

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[Glyphosate µM]0 62 12531 250 500

Mass Profiler Professional and Pathway Architect Identify the Target of Glyphosate

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EPSP Synthase, the target of glyphosate

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• Technical replicates (6x) of pooled biological samples treated with three concentrations levels of drug were analyzed

• Replicates were analyzed interspersed across days of runs

• Data demonstrate consistent signal response and reproducible retention

Analytical Reproducibility Enables “Hands Off” Quantitation

Metabolic Changes Include Local Effects and Characteristic Global Responses - a Fingerprint of Mode of Action

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Summary

Metabolomics dMRM Database and Method provides an optimized, robust method for routine targeted analysis of more than 215 central carbon metabolites

Agilent offers an comprehensive metabolomics portfolio that includes sample preparation, data acquisition and data analysis

June 8, 2016

Confidentiality Label

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Acknowledgements

University of Toronto- Adam Rosebrock

• Soumaya Zlitni

- Amy Caudy

McMaster University- Eric Brown

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DonnellyCentrefor Cellular + Biomolecular Research

simplifying the routine lc/ms analysis of central carbon ...€¦ · simplifying the routine lc/ms...

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