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Tandem mass spectrometry analysis of prostaglandins and

isoprostanes

Jeevan K Prasainjprasain@uab.edu6-2612

Overview

• Introduction to PGs and their synthesis

• Mass spectrometry characterization of PGs and isoprostanes

• PGs in Cox-dKO pups and C. elegans

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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.

Polyunsaturated fatty acids (PUFAs)-substrates for PGs

Arachidonic acid (AA)Omega-6

Eicosapentaenoic acid (EPA)Omega-3

Dihomo-y-linolenic acid (DGLA)Omega-6

Series 1

Series 2

Series 3

HO

HO

COOH

OH

PGF2alpha

HO

HO

COOH

OH

HO

HO

COOH

OH

PGF1alpha

PGF3alpha

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Cox-dependent PGs synthesis

R1O

O

O P

OH

OO

N

R2

O

O

Phospholipase A2

Arachidonatemoiety

Enzymatic- cyclic pathways

Release first

NSAIDs

lipoxygenaseleukotriene

Dietary linoleic acid (C18: ∆9,12)

elongase

desaturase

Arachidonic acidC20: ∆5,8,11,14)

OOH

COOH

PGG2

O

O

OH

COOHO

O

PGH2

Non-enzymatic isoprostane synthesis

-H•COOH

arachidonic acid (20:4n-6)

COOH

OO

COOHO

O

COOHHO

HOOOH

OH

15

O2

Cyclization

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Structural representation PG based on ring features

R = aliphatic chain

Prostaglandin analysisConcentration range nM-pM in biological samples

1. Immunoassay (poor specificity for isomeric PGs, and only one or a few compounds/assay)

1. GC-MS (derivatization needed)

1. LC-MS/MS

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Inte

nsity, cp

s

m/z, amu40 100 160 220 280 340

3.8e6193.1

309.2164.9

171.2 291.1208.9

247.2191.1111.2

353.4263.1

255.0173.0

229.2273.2

137.1 181.2281.2

219.043.2 113.0

124.8 299.2

335.159.3 138.8

ESI-MS/MS of the [M-H]- from PGF2 m/z 353 using a quadrupole mass spectrometer

-44 Da

O-

O

OH

HO

HO

Fragmentation scheme of PGF2 [M-H]- m/z 353

Ions m/z 309, 291, 273 and 193 are indicative of F2-ring

Adopted from Murphy et al. Analytical Biochemistry, 2005

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Source: Murphy et al. Analytical Biochemistry, 2005

What information does deuterium labeling at C-2 and C-3 ofPGF2 provide us for structure elucidation of PG?

6 9 12 15 18 90.0

3.0e5

Inten

sity, cps

11.85 12.65

14.09

12.40

PGF2a

PGD2

PGE2

Lipoxin B4 PGJ2

PGD1

Separation of PGs[A] and standard curve of PGF2alpha [B]

1.0e-2 1.0e-1 1.0e0 1.0e1 1.0e2

1.0e3

1.0e4

1.0e5

1.0e6

Area, co

un

ts

[B]

[A]

Concentration, ng/mL

r = 0.9969

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MS/MS fragmentation of PGE2 and PGD2 m/z 351.00

m/z, amu

20 80 140 200 260 320

1.9e6

Inten

sity, cps

189.0

271.2

108.9112.9 135.0 162.9 217.1174.8 203.9158.967.0 191.0

269.3 333.3315.0170.9119.0 184.981.1 157.958.9

20 80 140 200 260 320

2.1e6

Inten

sity, cps

189.1

271.1

203.059.1 135.0106.981.2 191.0 269.3 315.1131.6

PGD2, Rt 12.4 min

PGE2, Rt 12.07 min

The first loss of water, m/z 189 and m/z 233 arecharacteristics of PGE2/PGD2

MS/MS fragmentation of PGE2 [M-H]- m/z 351

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Deuterated PG standards are used for quantitativeanalysis of PGs in a extract

Source: Cao et al. Analytical Biochemistry, 2008

15(R)-Prostaglandin F2α

Prostaglandin F2α

8-iso Prostaglandin F2α

8-iso-15(R)-Prostaglandin F2α

min

PGs and diastereoisomer isoprostanes can be distinguished based on retention time in LC-MS

Prasain et al., J Chrom B. 2013

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1 4 7 10 13 16 190

650

Intensity, cps

1.44

min

Prasain et al., J Chrom B. 2013

SRM chromatogram showing isoprostanes and PG in an AKI patience

Cox-independent PGs

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C. elegans culture, lipid extraction and mass spectrometry analysis

Hex

ane

Lipid

Aq

. ac

eto

ne

Homogenate+ IS

Aci

difi

ed

Aq/

ace

ton

eC

HC

l 3la

yer

Centrifuge

Hexane

3 M HCOOH+ CHCl3

ConcentrationCHCl3 layer

Reconstituted 80% MeOH

HPLC autosampler

Worm culture

LC-MS/MS/MS

CollectionStored -80 0C

Grown with NA22 E. Coli bacteria

Prasain et al., JoVE, 2013

WT

mutant

Product ion

Survey scan

MRM

McKnight et al., (Science, 2014)

Cox-independent PGs is widespread

Great similarities between C. elegans and Cox dKO pups PGF2 profile

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353

100 130 160 190 220 250 280 310 340

4.2e5

Inten

sity, cps

193.1

165.0 247.2

353.4263.2209.1

111.1 291.1 309.3170.7

273.0191.0181.2

255.3126.8

281.10112.8229.1163.2

299.2235.3147.2335.4

197.3

[A] PGF2 alpha, [M-H]-

m/z 353 MS/MS

110 140 170 200 230 260 290 320 350

1.9e4

Inten

sity, cps

273.0193.1 220.8 309.2

291.5

167.0251.4208.6

168.8157.2

247.6335.0234.8

[B] CePGF2 alpha, [M-H]-

m/z 353 MS/MS

LC-MS/MS of ion m/z 353 [M-H]- from wild type C. elegansextract confirmed that CePGF2 is a PGF2alpha-like PG

Edmonds et al., Dev Cell. 2010

Mass/Charge, Da

353.2318

354.2397

60 100 140 180 220 260 300 3400

250

309.2063

193.1230291.1961165.1265

247.2056193.1586171.1002

111.0825 335.2240

317.2148

273.2187209.119383.0528 235.1307

219.1748

146.938959.0178

255.2152

Std. PGF2alpha

C2H4O44.02 da

44.02 da

High-resolution mass spectrometry analysis of PGF2alpha

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0.5 2.0 3.5 5.0 6.5 8.00

2000

Inte

nsity, cp

s

4.73

Ent-PGF2alpha

0.5 2.0 3.5 5.0 6.5 8.00

4400

Inte

nsity, cp

s

5.17

PGF2alpha

Separation of PGF2alpha and its enantiomer onlypossible in chiral normal phase column

(ChiralPak AD-H column) APCI –ve ion mode

Hoang et al., PLOS Genetics. 2013

Hoang et al., PLOS Genetics. 2013

Cox-independent PGF2 showed close similarity with ent-PGF2a in chiral normal phase LC-MRM

Cox-independentPGF2 in C. elegans

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Conclusions

• Based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), genetic analyses, and bioactivity assays, C. elegans synthesizes Cox-independent 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.

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