Tandem mass spectrometry analysis of prostaglandins and ...
Transcript of Tandem mass spectrometry analysis of prostaglandins and ...
3/20/2020
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Tandem mass spectrometry analysis of prostaglandins and
isoprostanes
Jeevan K [email protected]
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
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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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