1. BIOM 209/CHEM 210/PHARM 209 Lipid Cell Signaling
Genomics, Proteomics and Metabolomics
January 5, 2016
Professor Edward A. Dennis
Department of Chemistry and Biochemistry
Department of Pharmacology, School of Medicine
University of California, San Diego
Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or individual slides (without alteration) for academic, non-profit and non-commercial purposes. Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial”
E.A. DENNIS 2016 ©

2. OMICS OVERVIEW: GENOMICS/PROTEOMICS/METABOLOMICS
OF LIPID METABOLISM AND CELL SIGNALING
AND IMPLICATIONS FOR HUMAN DISEASE PROFILING AND BIOMARKER DISCOVERY
A. LIPID MAPS Initiative in Lipidomics B. Human Plasma Lipidome C. Eicosadomics of Macrophages D. Genomics and Proteomics Integration

3. Lipid Metabolites And Pathways Strategy GENOMICS (TRANSCRIPTOMICS)
LIPID MAPS
Lipid Metabolites And Pathways Strategy
GENOMICS
(TRANSCRIPTOMICS)
4 base side chains
~25,000 coding genes
PROTEOMICS
20 amino acid side chains
~30,000 in databases
METABOLOMICS
nucleic acids, amino acids, sugars and fats: >105
~42,000 in databases
“LIPIDOMICS”
all the fats: >105
~40,000 in LM database
Dennis (2009) Lipidomics Joins the Omics Evolution, PNAS, 106, 2089.

4. Increases in Omics Citations 1985-2009
Number of Citations
Wenk MR (2010) Lipidomics: New Tools and Applications. Cell, 143,
Year

5. Lipidomics Publications 2002-2015

6. Human Plasma Lipidomics
NIST collected (pooled) fasting plasma from 100 individuals
50% female and 50% male; age 40-50
15% of the total taken from individuals of Hispanic origin

7. Human Plasma Metabolites (mg/dL)
Lipids
Nucleic Acids
Amino Acids
Sugars

8. Human Plasma Lipid Categories (M)
Sterol Lipids
Fatty Acyls
Sphingolipids
Glycerolipids
Prenols
Glycerophospholipids

9. Lipid categories and Species in the Human Plasma *SRM
Lipid Category
Number of Species
Sum (nmol/ml)
Sum (mg/dl)
Fatty Acyls
107
214
5.8
Glycerolipids 74
1110
93.7
Glycerophospholipids
160
2590
200
Sphingolipids
204
318
23.7
Sterol Lipids 35
3174
123
Prenol Lipids 8 5
3.7
Total
588
7411
449.9
J Lipid Res 51, (2010)
*SRM = standard reference material

10. SRM: Prostaglandins, Isoprostanes

11. SRM: Sterols

12. SRM: Cholesteryl Esters

13. SRM: Phosphatidylethanolamines

14. Human Plasma Lipid Diversity
J Lipid Res, 51, (2010)

15. Plasma Lipids in the Metabolic Syndrome
Quehenberger & Dennis, New Eng. J. Medicine, 365, (2011)

16. Implications of Lipidomics for the Future of Clinical Medicine
Identification of metabolites in human plasma and other tissues for diagnostic purposes
Discovery of novel metabolites as biomarkers for disease states
Quantitation of metabolites which permit dynamic monitoring of disease pathophysiology over time
Evaluation of the efficacy of pharmacotherapeutic agents targeted to specific diseases which affect lipid metabolic pathways (statins)

17. NIGMS Large Scale Collaborative Grant
LIPID MAPS
NIGMS Large Scale Collaborative Grant
“Glue Grant” [NIH U54 GM 69338]
Mouse Macrophage: RAW cell line & primary cell
Environmental Agonist: initially LPS, then oxidized LDL
HPLC/Mass Spectrometer: identify known & new, quantify
Synthesis/Characterize: new lipids, MS quantitative standards
Bioinformatics: informatics and lipid networks
Website: LIPID MAPS -- Nature Lipidomics Gateway,
LIPID MAPS TM

18. “CLASS”: Comprehensive Lipidomics Analysis using Separation Simplification
cells or tissues
probe
cells
tissues
medium
a “divide-and-conquer” strategy
sonicate
homogenate
category specific
internal standards
(deuterated, odd-chain carbon)
category optimized
extraction
(liq-liq, SPE)
extract
category specific GC LC
(GC, NP-HPLC, RP-HPLC, chiral, specialty)
mass spectrometer
(variables)
Harkewicz & Dennis, “Applications of mass spectrometry to lipids and membranes”
Ann Rev Biochem, 80: (2011)
1. Mass spectrometer types
2. Ionization mode
3. Additives (for ionization)
4. Mass spectrometer monitoring modes

19. Kdo2-Lipid A (KLA, LPS subspecies) A specific agonist of TLR-4 on RAW macrophages Raetz et al., 2006, J. Lipid Res. 47: 1097
Nuclei – DAPI
Mitochondria – MitoTracker Red
O-Specific chain
Polysaccharide
Core
Kdo
Glycophospholipid
Lipid A 19

20. Dennis et al (2010)
J. Biol. Chem, 51,

21. Phospholipase A2 (PLA2) Function in Arachidonic Acid ReleaseAA PX FA HO PX FA
Arachidonic Acid
Cyclooxygenase
Lipoxygenase
Aspirin
NSAID
Prostaglandins
Leukotrienes
Dennis et al (2011) Chem Rev, 111,

22. Eicosanoid Signaling Pathways in RAW264.7 Macrophage
ATP
ATP
LPS
(KLA)
Numerous
eicosanoid
metabolites
Buczynski et. al. (2007) JBC, 282, 22834

23. Basic Macrophage Experimental Scheme
Eicosanoid enzyme
mRNA & protein
Macrophages
± Kdo2 Lipid A
(TLR4)
± 2 mM ATP
(P2X7)
Solid Phase Extraction
& LC-MS/MS
Isolate Media &
Solid Phase Extraction
(Eicosanoids)
LC-MS/MS

24. Cellular Eicosanoid Metabolism
Buczynski, Dumlao, Dennis (2009) JLR, 50, 24

25. Cellular Eicosanoid Metabolism
COX
CYP(ω)
5-LOX
15-LOX
CYP(EET)
12-LOX
Buczynski, Dumlao, Dennis (2009) JLR, 50,

26. Cellular eicosanoid metabolism
CYP(w)
COX
5-LOX
CYP(EET)
15-LOX
12-LOX
Buczynski, Dumlao, Dennis, 2009, JLR, 50:

27. Cellular eicosanoid metabolism
COX

28. KLA / RAW Macrophage: Time-course
Extracellular
Intracellular
PGD2 + metabolites
(Extracellular)
Buczynski et al (2007)
JBC, 282, 22834

29. Fluxomics: KLA / RAW Macrophage Time-course
Gupta, Maurya, Stephens, Dennis, Subramaniam (2009) Biophys J, 96, 4542

30. Good fit! Prediction of Eicosanoid Fluxes in
TLR4-Primed/Purinergic-Stimulated BMDM
COX pathway
LO pathway
This is a result of prediction of eicosanoid profile in KLA-primed ATP-stimulated BMDM cells.
For most time points, the difference between the simulated and experimental data under treatment and control conditions was within the standard error of the mean.
Good fit!
Kihara et al, (2014) Biophys J, 106,

31. Macrophage Phenotypes
Resident Peritoneal (RPM)
Thioglycolate-Elicited Peritoneal (TGEM)
Sterile Inflammation
Bone Marrow-Derived (BMDM)
RAW264.7 Cell Line (RAW)
Tumor
M-CSF
Ab-MuLV

32. Eicosanoid Changes by Phenotype
8 Hr
PGI2
PGE2
Fold Increase
PGD2
TxB2
104 Eicosanoids analyzed
Fold Decrease
Not detected

33. COX-2 Metabolites and Transcripts

34. PGE2/PGD2 vs PGES/PGDS Transcripts

35. Proportionality of Eicosanoids and Transcripts
RPM
TGEM
BMDM
RAW
J. Leukocyte
Biology, 90, (2011)

36. Directed Proteomics on Enzymes of Eicosanoid Biosynthesis
Sabido, Quehenberger
Dennis, Aebersold (2012)
Mol Cell Proteomics 11: M

37. Protein Abundance after KLA Stimulation

38. Eicosanoid Genes to Proteins to Metabolites
Quehenberger & Dennis, New Eng. J. Medicine, 365, (2011)

39. THE NEED FOR “METABOLOMICS”
“Premiums in the shape of sensational discoveries may be hoped for, but cannot be assured even to the greatest genius. But what has to penetrate, relative to this question, more completely into the consciousness of pathologists, is this, that to understand zymoses, to be able to counteract them by rational, as distinguished from empirical or accidentally discovered means, is only possible by the aid of a complete knowledge of the chemical constitution of all the tissues, organs and juices of the body, and of all their possible products.”
Johann Ludwig Wilhelm Thudichum ( )
A Treatise on the Chemical Constitution of the Brain (1884)
Quoted from
[Joseph Needham (1971) The Chemistry of Life, Cambridge Univ Press p. 199]
“Premiums in the shape of sensational discoveries may be hoped for, but cannot be assured even to the greatest genuis. But what has to penetrate, relative to this question, more completely into the consciousness of pathologists, is this, that to understand zymoses, to be able to conteract them by rational, as distinguished from empirical or accidentally discovered means, is only possible by the aid of a complete knowledge of the chemical constitution of all the tissues, organs and juices of the body, and of all their possible products.
LIPID MAPS TM