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Eicosanoids Richard Harkewicz LIPID MAPS LIPID MAPS Lipidomics Workshop April 18, 2009 TM www.lipidmaps.org Eicosanoids Richard Harkewicz Departments of Pharmacology, Chemistry and Biochemistry The University of California, San Diego La Jolla, CA Other LIPID MAPS Eicosanoid Core Members: Aaron Armando Matthew Buczynski Edward A. Dennis Alexander Andreyev Darren Dumlao (Core Director) Joshua Brooks Oswald Quehenberger 1 Outline A) Brief description of eicosanoids B) Sample preparation/extraction C) Analytical methodology: LC-MS D) Library of eicosanoid standards E) Chiral chromatography – enzymatic or non-enzymatic? F) GC-MS methodology for free fatty acids G) DIMPLES/MS: A stable isotope substrate labeling strategy enabling the search for novel eicosanoids H) Comparison of LIPID MAPS eicosanoid approach with others in literature I) Future plans contact: [email protected] 2 stimulation release of (LPS or phospholipase A2 membrane phospholipid (PLA ) Kdo2-Lipid A) 2 (R = choline or ethanolamine) (outside the cell) O (inside (PLA2) R O P O CH2 the cell) OH CH O arachidonate COOH H2C O sterate (20:4) (18:0) free eicosatetraenoic acid (from the Greek eicosa for 20) AKA arachidonic acid (20:4, n=6) Eicosanoids – powerful mediators involved in inflammation that are derived from arachidonic acid and act in autocrine and paracrine fashion (signal at or immediately adjacent to their site of synthesis) Once they are made, they are quickly secreted from the cell Transcription factor: Can also enter cell’s nucleus, binding and activating nuclear receptors Sometimes referred to as autocoids or local hormones 3 HO COOH COOH O COOH O TXA O HO OH 2 HO OH COOH PGF2α (vasoconstriction, smooth muscle O PGD HO PGI OH 2 (vasodilation, OH 2 contraction) hypotension) COX OH OH OOH COOH COOH COOH LTB4 (vasodilation, leukocyte OH 5-LO 5-HpETE adhesion, chemotaxis) AA COOH CONHCH2COOH 15-LO S LTD NH2 4 OH OH COOH COOH COOH HO HO (vasodilation, chemotaxis, OH LXA OOH 15-HpETE OH 4 leukocyte activation) LXB4 4 sample preparation and extraction 0 h 24 h 48 h LC-MS Solid phase analysis Plate LPS or Collect extraction Medium Cells Kdo2-Lipid A [100 ng/ml] 1. Solid phase extraction (Phenomenex® Strata-X 8B-S100-UBJ) 1. Spike medium 1. Plate cells with deuterated 2. Prewash SPE column 24 h internal standards with 2 ml MeOH 2. Stimulate (10 ng/100 μl) then 2 ml H2O (LPS or Kdo) 3. Apply sample to column 2. Add EtOH to 24 h then wash with 2 ml 10% obtain 10% EtOH MeOH 3. Remove medium medium solution (≈2 ml per well) 4. Elute eicosanoids with (≈2e6 cells per well) 3. Centrifuge 5 min 1 ml MeOH @ 3000 rpm 5. Dry under vacuum and resuspend in 100 μl HPLC solvent A 5 column Solvent A RP HPLC Gradient 80 Vydac® 201TP52 H20/ACN/formic acid: 2.1 mm X 250 mm 63/37/0.02 60 Solvent B Flow rate 300 μl/min ACN/IPA: 50/50 40 solvent A solvent B Solvent B (%) 20 LC pump 0 0 5 10 15 20 25 Time (min) auto RPLC C column ABI Analyst sampler 18 collision software gas (N2) (­)ESI CEM detector Q1 mass q2 Q3 linear analyzer collision trap mass cell analyzer Applied Biosystems® 4000 QTrap Quadrupole mass spectrometer 6 Applied Biosystems Analyst 1.5 Eicosanoid Standard “scheduled MRM” allowing >1000 MRM pairs Mixture fragment LC/MS - MRM isolate precursor precursor isolate product Multiple Reaction Monitoring “specialized” form of MSMS ) 1 27 ) Q1 q2 Q3 2 / 1 E 7 G 2 P 351 9) ( 2 / 6 D 1 1 G 5 / P 3 MRM pair: + 100% 9 ( 36 HETEs ( (hydroxyeicosatertraenoic great S/N & 2 B acids) comprehensive X 75% T ) 3 9 ) 1 / 9 3 203) 5 5 2 50% A / Rel. Intensity 3 ( A 3 335/ ( 0 α 3 2 ( F 4 G B P T 25% L 2 4 6 8 10 12 14 16 Time, min T 7 M COOH The HETEs OH COOH CH3 HO 11(s) HETE 8(s) HETE (319/155) (319/167) CH3 5(s) HETE (319/115) OH COOH COOH CH3 CH3 12(s) HETE OH (319/179) 15(s) HETE (319/113) COOH CH3 OH 10.8 11.0 11.2 11.4 11.6 11.8 Time, min 8 Library of Eicosanoid Standards (>200) (http://www.lipidmaps.org) Provides: Chemical structure in ChemDraw® format LC and MS protocols MSMS fragmentation spectra LC retention times for given set of conditions Web-link to Cayman Chemical for each eicosanoid The eicosanoid library provides information from which comprehensive methods T can be created and used to survey eicosanoid release from stimulated cellsM 9 use of deuterium labeled internal standards allows absolute quantitation for a number of eicosanoids D D O - O - COO COO D D HO HO OH OH - PGE -d4 [M-H]- = 355 PGE2 [M-H] = 351 2 example of internal standard calibration curves used for quantitation PGE 12,14 2 15d- D PGJ2 5-HETE MRM: 351/189 MRM: 315/271 MRM: 319/115 100 100 100 r ² = 0.9995 r ² = 0.9975 10 10 10 r ² = 0.9993 1 1 1 0.1 MRM Ratio 0.1 0.1 0.01 0.01 0.01 0.001 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 Analyte (ng) 10 chiral chromatography aids in determining enzymatic vs. nonenzymatic origin atmospheric pressure chemical column ionization (APCI) used Flow rate Chiralpak® AD-H 500 μl/min 4.6 mm X 250 mm Kdo2-Lipid A stimulated Kdo2-Lipid A stimulated RAW cells RAW cells 10.24 Solvent A Solvent B 8.85 MRM: MRM: 9.32 hexane/EtOH/H20/formic acid: EtOH : 100 319/167 319/113 96/4/0.08/0.02 NP HPLC Gradient 50 40 7.6 8.4 9.2 10.0 8.6 9.4 10.2 11.0 Time, min Time, min 30 10.27 9.34 11(S) HETE 15(S) HETE STD 15(R) HETE STD 20 11(R) HETE STD Solvent B (%) STD 9.35 10 8.87 0 010203040 7.6 8.4 9.2 10.0 8.6 9.4 10.2 11.0 Time (min) Time, min Time, min 11 GC-MS analysis of free fatty acids Pentafluorobenzyl (PFB) derivatives of the free fatty acids are formed and negative chemical ionization employed. Produces intact molecular anions - optimal for quantitation (using deuterium labeled standards). Agilent 6890N gas chromatograph Agilent 5973 single quadrupole mass analyzer Selected ion monitoring (SIM) mode monitoring the [M-H]- anions. analysis of essential fatty acids in macrophages Quehenberger et al. (2008) Prostaglandins Leukot. Essent. Fatty Acids 79 pp. 123-129 12 Are biologically significant eicosanoids being overlooked? Are there species for which we have no prior knowledge of or expectation of their presence and, hence, no available MRM pairs required for their detection? To address these concerns a mass spectral based stable isotope labeling strategy has been developed DIMPLES/MS: Diverse Isotope Metabolic Profiling of Labeled Exogenous Substrates using Mass Spectrometry Harkewicz et al. (2007) J. Biol. Chem. 282 pp. 2899-2910 Incubation of cells in medium supplemented with deuterium-labeled arachidonic acid (AA-d8) D D D D COOH COOH AA D D D D AA-d8 13 uptake and incorporation of AA-d8 by cells 0 h 24 h 48 h LC-UV-MS solid phase analysis plate Kdo2-Lipid A collect extraction cells [100 ng/ml] medium add AA-d8 [12.5 μg/ml, 40µM ] exchange of the PL’s endogenous sn2 fatty acids for the exogenous AA-d8 100 % (R = choline or ethanolamine) O AA-d8 AA R O P O CH2 60 % OH CH O arachidonate H2C O sterate 20 % AA-d8 Remaining in Media (%) 0 5 10 15 20 AA-d8 Incubation Time (h) 14 so as not to be a AA/AA-d8 mass spectral doublet targeted analysis indicative of eicosanoid: full-scan MS (1) AA origin 10% serum (fetal calf) 2) upregulated by Kdo AA-d8 supplemented 357 D D D D [(PGD + d6) - H]- 2 COOH 351 - [PGD2 -H] D D D D AA-d8 358 - COX [(PGD2+ d5) - H] - [(PGD2+ d7) - H] + PGD-synthase Rel. Intensity (%) Rel. Intensity 356 OH D D D D 352 COOH 359 355 353 O D D HO D PGD2+ d7 351 353 355 357 359 m/z, amu 15 observed production of a class of dihomoprostaglandins Harkewicz et al. (2007) J. Biol. Chem. 282 pp. 2899-2910 Kdo – Lipid A stimulated RAW cells HO dihomo-PGD2 2 O dihomo-PGE2 Control (NO AA-d8) COOH COOH AA-d8 supplemented O HO OH OH 385 COOH 385 - [(M+d6) - H]- [(M+d6) - H] - m/z = 331 [M-H] [M-H]- 379 adrenic acid (22:4 n-6) 379 339 384 386 100% 386 384 380 380 381 383 387 [(22:4 n-6) + d8 – H]- 381 383 387 HO dihomo-PGF2α dihomo-11(R)HETE COOH COOH HO 389 HO - 347 [(M+d8) - H]- OH [(M+d8) - H] [M-H]- 354 355 [M-H]- [(22:4 n-6) – H]- 388 381 353 390 348 387 356 331 352 357 382 383 386 349 12,14 12,14 dihomo-∆ -PGJ dihomo-15d-Δ -PGD2 Rel. Intensity 338 2 HO COOH COOH O OH 367 O 367 340 [(M+d6) - H]- 332 337 [M-H]- [(M+d6) - H]- [M-H]- 333 361 361 366 368 368 366 362 362 365 369 363 365 369 331 334 337 363 m/z, amu 16 another approach to eicosanoid lipidomics and the search for novel eicosanoids Serhan Lab developing theoretical databases and algorithms based on virtual UV absorption, tandem mass spectrometry and LC retention times for identifying potential eicosanoids .
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