metabolism platforms - cpsa-metabolomics.comcpsa-metabolomics.com/2016/vendor_pirman.pdf · –...
TRANSCRIPT
Metabolism platforms
Agios Pharmaceuticals
• Metabolome provides opportunity for novel therapeutics in cancer and rare genetic disease
• Personalized targeted therapies
• Apply metabolism studies at very early stage in each programs (establish early stage biomarkers) – Untargeted metabolomics, fluxomics,
mutli-omics
– Translational research
– Target ID and validation
– Patient stratification
• 2-hydroxyglutarate in IDHm cancers
• Ex vivo glycolytic flux measurement in pyruvate kinase disease patients
WT PKD
2,3-‐DPG
ATP Primary metabolic consequences are
known
Deep, quan?ta?ve understanding of affected pathway
Metabolic consequences are unknown
Specific, Translatable human blood
Layers of valida?on in vitro
and in vivo
Secondary consequences of lesion, & modula4on with target engagement are
unknown
Analytical approaches to understanding metabolism in disease
Biology and metabolism: a symbiotic relationship
• In vitro, in vivo, pa4ent samples
• Model development • Target valida4on
Biology
• Mul4-‐omics plaBorms • Metabolism, lipidomics, metabolomics
• Targeted assays • Biomarker iden4fica4on
Analy4cal • Automated data analysis • Metabolomics (targeted and untargeted)
• Flux Analysis • Pathway analysis
Informa4cs
Agios Metabolism Platform: Tools and Technology Automation and Mass Informatics Workflows (scale reflects effort)
proteomis
Metabolite and Lipid Extrac6on & Harvest robo6cs
Integrated so<ware, analysis
Metabolite-‐Centric Data Processing
Visualiza6on, data analysis, Informa6cs tools
Automated data analysis and informa6cs workflows
Mass Spectra
Data QC, Correc6on, Transforma6on
Hypothesis genera6on
Kine6c Flux Labeling
Transcriptomics, Proteomics, Lipidomics…
Isotope Tracing
MS
Metabolomics
• 3 QEs • Xevo TQS • AB 4000 • Thermo GC-‐QQQ
• Fusion • Agilent QTOF
Agios Metabolism Platform: Tools and Technology
ü High mass resolu4on and accuracy ~ 20ppm ü Fast rela4ve quan4ta4on (1 min/assay)
ü Broad & untargeted coverage of intracellular small molecules
ü (2000-‐4000 ions detected per sample)
FIA – QToF Profiling Q Exac?ve LCMS
Agilent 6500 QTOF
Lipidomics +/-‐ mode
Metabolomics -‐ mode + mode
ü High mass resolu4on and accuracy ~ 5ppm ü Both targeted and untargeted broad profiling
ü UPLC separa4on of analytes for ID confirma4on
Library of ~700 metabolites annotated with m/z and RT
HRAM spectra on a QExac?ve at 140k resolu?on
15N
Neutron
13C
Δm = 0.0063 amu arginine
LC-MS/MS Lipidomics
Waters iClass Aquity w/ Thermo QExac4ve
Extract HRMS+MS/MS (pos/neg)
Signals automa4cally
annotated using MS/MS and assembled for
sta4s4cal analysis
Observed >800 lipids from biological samples • Phosphoglycerolipids • Glycerolipids • Sphingolipids • Cholesterol esters • Free fady acids
Limited coverage on sterols, prenols, polyke7des
Comprehensive data analysis/interpreta4on to iden4fy metabolic perturba4ons
Analy4cal plaBorm
R e te n tio n T im e (m in )
Ion
In
ten
sit
y
0 1 0 2 0 3 00
5 .0´1 0 8
1 .0´1 0 9
1 .5´1 0 9
2 .0´1 0 9
2 .5´1 0 9
LPC (int std)
PC, PE, SM
TAGs
1 2 3 4 5
Summary of platforms
Proteomics
NEW!
ThermoFusion
PTM analysis
Combine data
Lipidomics
QExactive
LC-MS/MS based
Identify >800 lipid species in 30 m analysis
Building informatic toolbox to interpret data (pathway mapping)
Metabolomics
QQQ, QToF, QExactive
LCMS + GCMS (NEW!)
Identify ~600 metabolites from known library
Isotopic labeling studies
Informa4cs
Informatics analysis
• Combi-Omics – Metabolism-transcript
– Protein-transcript
– Metabolism-protein-transcript
• Flux analysis
• MIDA analysis
• Automated workflows – FIA-TOF
– Peak annotation
– Statistical characterization
Gaps in the process
• Agios process – Experiments design
(BiologyàLIMS)
– Execution (mostly automated)
– Conversion to mzXML (auto)
– Peak ID via MAVEN (manual or auto)
– Nat. abundance isotope correction, quantitation (custom software)
– Flux analysis, data visualization, multi-omicsàmetadata preserved (SpotFire)
• Gaps – LIMS and metadata
• Customize older LIMS softwares
– Vendor neutral peak picking software to identify stable isotope labels
• Maven works, but can be slow with automatic workflow
– Lipid pathway analysis tools (not well annotated in KEGG)
– Multi-omics pathway analysis
– Data repository of unknowns