Tandem mass spectrometry analysis of prostaglandins and isoprostanes Jeevan Prasain jprasain@uab.edu 6-2612 verview Introduction to PGs and their synthesis Mass spectrometry characterization of PGs and isoprostanes PGs in Cox-dK pups and C. elegans 1
Prostaglandins Derived from 20 carbon PUFA, have short half-lives and act as local hormones Bind to specific cell surface G-protein coupled receptors and implicated in a number of physiological processes including reproductive function. NSAIDs acts through inhibiting Cox and hence PGs and exert various effects, including infertility. However, the genetics of prostaglandin synthesis and action have largely been unexplored in vivo. Mammalian systems are not well suited for discovering new genes and molecular mechanisms involved in PG action. The nematode C. elegans provides a platform for discovering roles of genes and mechanisms that would provide an ideal complement to mammalian systems. Cox-dependent PGs synthesis Dietary linoleic acid (C18: 9,12 ) elongase desaturase Arachidonic acid C20: 5,8,11,14 ) Steroids Phospholipase A2 R 2 Arachidonate moiety Release first Enzymatic- cyclic pathways R 1 lipoxygenase P N H leukotriene H CH NSAIDs PGG2 CH H PGH2 2
Non-enzymatic isoprostane synthesis CH Proton abstraction CH 2 arachidonic acid (20:4n-6) Cyclization H H 15 H CH H CH Structural representation PG based on ring features R = aliphatic chain 3
C. elegans culture, lipid extraction and mass spectrometry analysis Worm culture Grown with NA22 E. Coli bacteria Collection Stored -80 0 C Survey scan WT Homogenate + IS Centrifuge Hexane Hexane Aq. acetone Acidified Aq/acetone 3 M HCH Concentration + CHCl 3 CHCl 3 layer CHCl 3 layer LC-MS/ MS/MS Reconstituted 80% MeH Lipid HPLC auto sampler mutant Product ion MRM Prasain et al., JoVE, 2013 LC-MS/MS data summary for chemically synthesized F-series PG standards. Hoang et al., 2013 4
ESI-MS/MS of the [M-H]- from PGF2 m/z 353 using a quadrupole mass spectrometer 3.8e6 193.1 H - H H 309.2 164.9 171.2 291.1 208.9 191.1 247.2 111.2 229.2 263.1 124.8 299.2 173.0 255.0 273.2281.2 43.2 59.3 138.8 113.0 137.1 181.2 219.0-44 Da 335.1 353.4 40 100 160 220 280 340 m/z, amu Fragmentation scheme of PGF2 [M-H] - m/z 353 Ions m/z 309, 291, 273 and 193 are indicative of F2-ring 5
What information does deuterium labeling at C-2 and C-3 of PGF2 provide us for structure elucidation of PG? Source: Murphy et al. Analytical Biochemistry, 2005 Separation of PGs[A] and standard curve of PGF2alpha [B] 1.0e6 [B] r = 0.9969 3.0e5 [A] 11.85 12.65 PGD2 PGF2a Area, counts 1.0e5 1.0e4 1.0e3 1.0e-2 1.0e-1 1.0e0 1.0e1 1.0e2 Concentration, ng/ml PGE2 12.40 Lipoxin B4 PGD1 PGJ2 14.09 0.0 6 9 12 15 18 9 6
MS/MS fragmentation of PGE2 and PGD2 m/z 351.00 1.9e6 189.0 271.2 PGE2, Rt 12.07 min 108.9 112.9 135.0 162.9 217.1 67.0 158.9 174.8 191.0203.9 269.3 58.9 81.1 119.0 157.9170.9 184.9 315.0 333.3 20 80 140 200 260 320 2.1e6 189.1 PGD2, Rt 12.4 min 271.1 203.0 20 59.1 81.2 80 106.9 131.6 135.0 140 191.0 200 269.3 260 315.1 320 m/z, amu MS/MS fragmentation of PGE 2 [M-H] - m/z 351 The first loss of water, m/z 189 and m/z 233 are characteristics of PGE 2 /PGD 2 7
Deuterated PG standards are used for quantitative analysis of PGs in a extract Source: Cao et al. Analytical Biochemistry, 2008 PGs and diastereoisomer isoprostanes can be distinguished based on retention time in LC-MS 8-iso-15(R)-Prostaglandin F 2α Prostaglandin F 2α 8-iso Prostaglandin F 2α 15(R)-Prostaglandin F 2α min Prasain et al., J Chrom B. 2013 8
SRM chromatogram showing isoprostanes and PG in an AKI patience 650 0 1.44 1 4 7 10 13 16 19 min Prasain et al., J Chrom B. 2013 Separation of PGF 2 alpha and its enantiomer only possible in chiral normal phase column (ChiralPak AD-H column) APCI ve ion mode 2000 4.73 Ent-PGF2alpha 0 0.5 2.0 3.5 5.0 6.5 8.0 4400 5.17 PGF2alpha 0 0.5 2.0 3.5 5.0 6.5 8.0 9
Cox-independent PGs is widespread Great similarities between C. elegans and Cox dk pups PGF2 profile McKnight et al., (Science, 2014) LC-MS/MS of ion m/z 353 [M-H] - from wild type C. elegans extract confirmed that CePGF 2 is a PGF 2 alpha-like PG 4.2e5 193.1 [A] PGF2 alpha, [M-H] - m/z 353 MS/MS 165.0 247.2 209.1 263.2 291.1 309.3 111.1 170.7 353.4 273. 126.8 163.2 181.2 191.0 229.1 335.4 112.8 147.2 0 235.3 255.3 0 281.1 299.2 197.3 100 130 160 190 220 250 280 310 340 1.9e4 353 [B] CePGF2 alpha, [M-H] - m/z 353 MS/MS 273.0 193.1 220.8 309.2 168.8 247.6 291.5 157.2 208.6 234.8 251.4 335.0 167.0 110 140 170 200 230 260 290 320 350 10
Is CePGF2- PGF 2 alpha, co-eluting stereoisomer, ent- PGF 2 alpha or a racemic mixture? CePGF2 Close similarity with ent-pgf 2a in chiral normal phase LC-MRM Hoang et al., PLS Genetics. 2013 Conclusions Based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), genetic analyses, and bioactivity assays, C. elegans synthesizes Coxindependent sperm guiding F-series PGs from PUFA precursors. F-series PGs are synthesized in Cox-deficient mice, indicating the possible existence of similar mechanisms in other animals. 11