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Oscar Yanes Ph.D.
CEK/CIBEK, March 13th 2013
Metabolomics: Bridging Gaps in Systems Biology
Genomics
Transcriptomics
Proteomics
Metabolomics
High throughput Non-hypothesis driven Shotgun (proteomics)
Genomics
Transcriptomics
Proteomics
Metabolomics
High throughput Non-hypothesis driven
MASSIVE DATA!
Genomics
Transcriptomics
Proteomics
Metabolomics
NOISY MASSIVE DATA!
High throughput Non-hypothesis driven
Genomics
Transcriptomics
Proteomics
Metabolomics
- phenotype
+ phenotype
Vitamins, hormones, etc
Hepatitis
Why metabolites?
Thomas Willis (1621-1675) describes the sweetish flavor of urine in diabetes mellitus
Traditional Chinese doctors 2000--‐1500BC used ants to evaluate patient urine for high glucose
Technological advances
Newborn screening by tandem mass spectrometry
Untargeted Metabolomics
Changes to any metabolite(s) within the entire metabolome: • typically not hypothesis-driven • investigations into unknown mechanisms • often performed with a Q-TOF
Targeted Metabolomics
Focus on specific metabolites: • typically hypothesis-driven • effect of drug/disease on specific pathway • often performed with QqQ in MRM mode
Untargeted metabolomics flowchart
time
mass intensity
Chemical standard
Natural product
Database Search
Metlin HMDB KEGG
Accurate mass from TOF profiling
m/z
50 70 90 110 130 150 170 190 210 230 250 270 290 310
*
Match
Q-TOF
Hypothesis?
MS/MS
Experimental validation
Targeted metabolomics
Data analysis
Biological interpretation
Hypothesis
Biomarker discovery
List of metabolites differentially regulated
Pathway analysis Model construction Scientific literature
Disease vs. control
Validation
Mechanism
GeneSpring
QQQ
Experimental Validation
Targeted metabolomics Molecular biology techniques
Liquid-based MS
Tissue imaging MS
Nanostructure Initiator Mass Spectrometry (NIMS)
Immunohistochemistry Reverse Transcription-PCR Gene expression array Cell cultures Animal experimentation . . .
Marchetti V. et al. “Differential Macrophage Polarization Promotes Tissue Remodeling and Repair in a Model of Ischemic Retinopathy” Sci Rep. 2011;1:76.
Yanes O. et al. “Metabolic oxidation regulates stem cell differentiation” Nat Chem Biol. 2010 Jun;6(6):411-7
Panopoulos AD. et al. “The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming”. Cell Res. 2012 Jan;22(1):168-77
Dorell M. et al. “Antioxidant or neurotrophic factor treatment Preserves function in a mouse model of neovascularization-associated oxidative stress” J Clin Invest. 2009 Mar;119(3):611-23.
Vinaixa M. et al. “Metabolomics reveals reduction of metabolic oxidation in women with polycystic ovary syndrome after pioglitazone-flutamide-metformin polytherapy”. PLoS One. 2011;6(12):
Metabolomics implicates altered sphingolipids in chronic pain of neuropathic origin
Nature Chemical Biology 2012, 22;8(3):232-4
Rat model of neuropathic pain
experiments performed on animals three weeks after surgery
peripheral nerve injury model Chronic constriction injury (CCI, sciatic) Tibial nerve transection (TNT)
a ipsilateral dorsal horn (97%) b contralateral dorsal horn (1%) c ipsilateral dorsal root ganglia (2%) d ipsilateral tibial nerve (1%) e plasma (2%)
Rat model of neuropathic pain
Pathological alterations in the central nervous system after peripheral nerve injury
•
•
Demyelination in the dorsal root
Control Injury (demyelination)
Inoue et al., Nature Medicine 10, 712-718 (2004)
Transganglionic degenerative atrophy
Activation of astrocytes
•
Sphingomyelin-ceramide pathway
peripheral nerve injury
Sphingomyelin-ceramide pathway
peripheral nerve injury
Hypothesis: myelin catabolites in the dorsal horn affect nociceptive processing following peripheral nerve injury
Measuring the effect of DMS in vivo
DMS was administered to rats intrathecally and mechanical allodynia was measured using von Frey filaments
–PWT is paw withdrawal threshold measured using the up-down method of Dixon (1980)
Dorsal horn was stained with anti-glial fibrillary acidic protein (anti-GFAP) and 4’,6’-diamidino-2-phenylindole (DAPI)
Astrocyte activation in vivo
• GFAP is an intermediate filament specific to astrocytes • The expression of GFAP is upregulated in the neuropathic pain state • Intrathecally administered DMS induces astrocyte activation
Vehicle treated DMS treated
Therapeutic Metabolomics
Genomics Proteomics Metabolomics
Phenotype
Genotype
Inhibition of acid ceramidase with N-oleoyl ethanolamine (NOE)
NOE administration (intrathecal) significantly reduced adverse response to mechanical stimuli following peripheral nerve injury
NOE significantly reduced astrocyte activation in the dorsal horn following peripheral nerve injury
Dorsal horn was stained with anti-glial fibrillary acidic protein (anti-GFAP) and 4’,6’-diamidino-2-phenylindole (DAPI)
NOE treated vehicle
Inhibition of acid ceramidase with N-oleoyl ethanolamine (NOE)
Genomics
Transcriptomics
Proteomics
Metabolomics
- phenotype
+ phenotype
Genomics
Transcriptomics
Proteomics
Metabolomics Untargeted Non-hypothesis-driven
Targeted Hypothesis-driven
Targeted Hypothesis-driven
Targeted Hypothesis-driven
Bottom-up Omics Integration
Untargeted Metabolomics (Non hypothesis-driven)
Targeted metabolomics (Hypothesis-driven)
Targeted proteomics (Hypothesis-driven) PTMs analysis
Quantitative proteomics
(MRM analysis) MRM
Fluxomics
(MS and NMR)
Targeted Transcriptomics (Hypothesis-driven)
mRNA regulation epigenetics
Bottom-up Omics Integration
Tecnoparc
Mass Spectrometry LC/Q-TOF LC/QqQ GC/QqQ GC/QTOF GCxGC TOF MALDI TOF/TOF Orbitrap Velos Pro+ETD
NMR 600 MHz Bruker Avance III +cryoprobe+ScanJet 500 MHz Bruker Avance III+HR-MAS probe
Next-generation Sequencing Ion Torrent PGM
Microarrays Agilent’s DNA array platform
Centre for Omic Sciences
http://omicscentre.com/
SPAiN
FRANCE
Barcelona Reus
Scripps Center for Metabolomics and Mass Spectrometry Gary J. Patti Ralf Tautenhahn Gary Siuzdak
The Scripps Research Institute
Acknowledgements
Rovira i Virgili University
Toni Beltran Miguel Rodriguez Maria Vinaixa Sara Samino Xavier Correig
