lipidome analysis of the oleaginous...
TRANSCRIPT
Lipidome Analysis of the Oleaginous Microalga Nannochloropsis oceanica IMET1 During Oil Accumulation
Danxiang Han
Laboratory of Algal Biotechnology and Research Arizona State University
The 7th ABO Summit, Orlando, FL, Sept 30- Oct 27, 2013
What is Lipidomics?
Cellular Lipid Extract of a Biological Sample
Cellular Content of Individual Molecular Species of Lipids
Mass Spectrometry
“ to quantitatively describe dynamic and temporal alterations in the content and compositions of different
lipid molecular species, whcih occurred during physiological and genetic perturbations”
Applications of Lipidomics for Deciphering Glycerolipid Metabolism Network in Microalgae
LPAAT TAG
PAP DGAT GPAT Lyso-PA DAG G-3-P PA
Acyl-CoA Acyl-CoA Acyl-CoA
GPAT
Endoplasmic reticulum
Chloroplast
Acyl-ACP
Cytosol
Free FA Lipid body
(TAG)
Acyl-CoA
G-3-P
Lipid body (TAG)
LPAAT PAP
DGAT PA DAG
TAG TE
LC-FACS
PC
PG
CDS
PGPPS
MGDG MGD1 DGDG DGD1
SQDG SQD2
PDAT
UDP-Glu UDP-SQ CDP-DAG
PGP PGPS
Galactolipids Phospholipids
Lyso-PA
DAG
MGDG MGD1
SQD1
DGTS PI
CDP-DAG
PE
Choline (C)
PhosphoC
CDP-C CDP-E
PhosphoE
PECT PEMEAT CCT
Ethanolamine (E) CT/ET CT/ET CDS
Acyl-ACP
Phospholipids
BTA
B
PIS DAG-CPT/EPT
PDAT
PGP
PG
PGPS
PGPPS
Met
Ado-Met B
TAA
DGDG
+ +
+ +
+ +
+
+ +
-
- -
-
+ + + +
+ + + +
+
+ +
+ +
+ + + + +
+ + + +
Solvent Evaporation
+ + + +
+
Analyte Ions
Coulomic Explosion
Q1 CE Q3
Triple Quadruple Mass Analyzer
Power
+ -
Schematic of the Principle of QQQ Electrospray Mass Spectrometry
Phosphatidylethanolamine
Anionic Lipids ([M-H]-)
Phosphatidylglycerol Phosphatidylinositol Sulfoquinovosyldiacylglycerols
Monogalactosyldiacylglycerol Digalactosyldiacylglycerol Triacylglycerol Phosphatidylcholine
Electrically neutral Lipids ([M+X]+, X = Na, NH4, etc)
MS/MS Analysis of Molecular Species of TAG
2 x10
0
0.2
0.4
0.6
0.8
Counts (%) vs. Mass-to-Charge (m/z) 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975
+ Product Ion:1 (46.571-47.378 min, 146 scans) (872.7 -> **) 577.2
599.0 872.7
573.2 [M+NH4-R2COOH-17]+
[M+NH4-R1COOH-17]+
[M+NH4-R3COOH-17]+
[M+NH4]+
m/z of R1COOH =278 m/z of R2COOH =256 m/z of R3COOH =282
TAG 52:4
C52:4 = TAG18:1/16:0/18:3
C16:0
C16:1
C16:2
C16:3
C16:4
C18:0
C18:1
C18:2
C18:3
2D MS Analysis for Identification of TAG species from Algal Crude Lipid Extract (Neutral Loss Scanning)
Counts (%) vs. Mass-to-Charge (m/z)
m/z 853
m/z 853 m/z 853
Lipid MS scan Collision Energy (V) Products for Acyl Group Identification
PC Precursor scan of m/z 184 18 [M+Na-N(CH3)3-RCO2H]+
DGTS Precursor scan of m/z 236 40 [M+H-RCH=CO]+
PE Neutral loss of m/z 243 40 [M+H- C2H8NO4P-RCH=CO]+
MGDG Precursor scan of m/z 243 45 [M+Na-RCO2H]+
DGDG Precursor scan of m/z 405 70 [M+Na-RCO2H]+
SQDG Precursor scan of 225 35 [RCO2]-
PI Precursor scan of 241 45 [RCO2]-
PG Precursor scan of 227 35 [RCO2]-
Identification of Membrane Glycerolipids from Algal Crude Lipid Extract 2 x10
0
0.2
0.4
0.6
0.8
1
1.2
Counts (%) vs. Mass-to-Charge (m/z) 250 300 350 400 450 500 550 600 650 700 750 800 850
491.1 769.2
513.1
242.7 307.0
[M+Na-R1CO2H]+
[M+Na-R2CO2H]+
[C9H16O6+Na]+
MGDG 16:3/18:3
Quantitation of Glycerolipids in Algal Crude Lipid Extract
MRM: m/z 845.3 -> m/z 589.4
TAG 16:0/16:4/18:2
y = 0.5008x + 0.3255 R² = 0.996
0
1
2
3
4
5
6
0 2 4 6 8 10
Rela
tive
Resp
onse
(1
6:0/
18:1
/16:
0 :1
7:0/
17:0
/17:
0)
Relative Concentration (16:0/18:1/16:0 : 17:0/17:0/17:0)
TAG C50
y = 0.6694x + 0.0671 R² = 0.9997
012345678
0 2 4 6 8 10
Rela
tive
Resp
onse
(1
6:1/
16:1
/16:
1 : 1
7:0/
17:0
/17:
0)
Relative Concentration (16:1/16:1/16:1 : 17:0/17:0/17:0)
TAG C48
y = 0.9513x + 0.2812 R² = 0.9981
0
1
2
3
4
5
0 2 4
Rela
tive
Resp
onse
(1
8:1/
16:0
/18:
1 : 1
7:0/
17:0
/17:
0)
Relative Concentration (16:1/16:1/16:1 : 17:0/17:0/17:0)
TAG C52
y = 0.4832x + 0.5672 R² = 0.9921
0
1
2
3
4
5
6
0 2 4 6 8 10
Rela
tive
Resp
onse
(1
8:1/
18:1
/18:
1 : 1
7:0/
17:0
/17:
0)
Relative Concentration (18:1/18:1/18:1 : 17:0/17:0/17:0)
TAG C54
Calibration Curve
2x10
0
0
0
0
0
1
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
ISTD
LC-ESI/MS
Relative Response vs Relative Concentration
Counts (%) vs. Mass-to-Charge (m/z)
Counts (%) vs. Retention Time
3h
4h
6h
12h
24h
48h
72h
96h
Quantitation of Molecular Glycerolipid Species of Nannochloropsis oceanica IMET1 in response to N-deprivation
0
5
10
15
20
25
30
35
40
45
Cont
ent (
μmol
.g-1
DW
)
TAG Species
Nitrogen-replete
Nitrogen-depletion
00.20.40.60.8
11.2
14:0
/16:
1/16
:114
:0/1
6:0/
16:1
14:0
/16:
0/16
:016
:1/1
6:1/
16:1
14:0
/16:
0/18
:216
:0/1
6:2/
16:1
16:1
/16:
0/16
:216
:1/1
6:0/
16:1
16:0
/16:
1/16
:014
:0/1
6:0/
20:5
16:1
/16:
1/18
:116
:1/1
6:0/
18:2
16:1
/16:
0/18
:216
:0/1
6:1/
18:1
16:0
/16:
0/18
:216
:0/1
6:0/
18:1
16:0
/16:
1/20
:516
:0/1
6:1/
20:4
16:0
/16:
0/20
:516
:1/1
8:1/
18:1
16:0
/18:
1/18
:118
:1/1
6:0/
20:5
Cellular Content of TAG Molecular Species of N. oceanica IMET1
C18:1 and C18:2 are primarily associated with PC
Role of PC Acyl-editing in TAG Synthesis
P-C
18/16:x 18:2
P-C
18/16:x 18:1
P-C
18/16:x 18:3
P-C
18/16:x 18:3 PC Lyso-PC
Acyl-CoA (C18:0, C18:1)
Acyl-CoA (C18:1, C18:2) PLA2
TAG
LPCAT
-2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5
0 10 20 30 40 50
Time (Hr)
Fold
Cha
nge
(log2
N-/
N+)
mRNA of the Acyl-Editing Enzymes
PLA2-1
LPCAT PLA2-2
PLA2-3
* *
* * 0.01.02.03.04.05.06.07.08.0
N-repleteN-depleted
Cellular Content of Molecular Species of PC
Cont
ent (
μmol
.g-1
DW
)
0
5
10
15
20
25
30
35
40
45
Cont
ent (
μmol
.g-1
DW
)
TAG Species
Nitrogen-replete
Nitrogen-depletion
00.20.40.60.8
11.2
14:0
/16:
1/16
:114
:0/1
6:0/
16:1
14:0
/16:
0/16
:016
:1/1
6:1/
16:1
14:0
/16:
0/18
:216
:0/1
6:2/
16:1
16:1
/16:
0/16
:216
:1/1
6:0/
16:1
16:0
/16:
1/16
:014
:0/1
6:0/
20:5
16:1
/16:
1/18
:116
:1/1
6:0/
18:2
16:1
/16:
0/18
:216
:0/1
6:1/
18:1
16:0
/16:
0/18
:216
:0/1
6:0/
18:1
16:0
/16:
1/20
:516
:0/1
6:1/
20:4
16:0
/16:
0/20
:516
:1/1
8:1/
18:1
16:0
/18:
1/18
:118
:1/1
6:0/
20:5
Detection of EPA associated with TAG in N. oceanica IMET1 under N-deprivation Conditions
20:5
P 16:0 (14:0)
P-E
20:4 20:5
P-E
18:1 20:5
P-E
18:0 18:1
P-E
18:3 20:5
P-E
20:4 20:4
P-E
18:3 20:4
P-E
18:2 18:3
P-E
20:5 20:5
PE Pool (C18/C20 desaturation)
P-C
18:1 18:2
P-C
18:1 18:1
P-C
18:1 18:3
P-C
18:2 18:3
PC Pool (C18 Desaturation)
P-S
18:2 18:3 PS
18:3 -CoA
OH
18:2 18:3
DAG
Kenn
edy
Pa
thw
ay
OH
20:5 20:5
MGal
20:5 20:5
MGal
20:5 16:0 (14:0)
DGal
20:5 16:0 (14:0)
DAG MGDG
Chloroplast
MGDG DGDG
P-G
20:5 16:0
PG
PA
OH
20:5 20:5
OH
20:4 20:5
TS
20:4 20:5
TS
20:5 20:5
DAG
DAG DGTS DGTS
P
OH OH
C18:2 C18:3 18:2 -CoA
Δ12-DES Δ6-DES Δ6-DES
Δ5,6-DES Δ5,6-DES Δ5-DES Δ5-DES
Δ12, 6-DES Δ12, 6, 5-DES
Δ6-ELO Δ6-ELO
Δ6-ELO
Δ5,6-DES Δ6-ELO
PLA2
LC-FACS
MGD1
DGD1
PAP MGD1
BTAA
BTBB
Δ5-DES
PSD
PSS CDS PGPS PGPPS
Biosynthesis and Allocation of EPA in N. oceanica IMET1
MGDG DGDG PG PE DGTS
TAG PDAT (1)
Lipase (14/39) Fatty Acid Fo
ld C
hang
e (N
-/N
+)
0
0.5
1
1.5
2
0 10 20 30 40 50
Time (hr)
PSD1 mRNA
-0.20
0.20.40.60.8
0 10 20 30 40 50 60
Time (Hr)
PDAT mRNA
DGAT
ER
Chloroplast
Acetyl-CoA Acyl-ACP (C16:0, C18:0, C18:1)
Malonyl-CoA
Cytosol
Lyso-PA PA DAG TAG
Free FA
G-3-P
Acyl-CoA
Lyso-PA PA DAG TAG G-3-P DGAT PAP LPAAT GPAT
ACCase Type II FAS
DGTS, PE
MGDG,DGDG,PG PDAT
PDAT
SAD
PAP LPAAT GPAT
Pathways Involved in TAG Biosynthesis in IMET1
Eukaryotic Kennedy Pathway
LysoPC PC PC Acyl Editing Cycle
Prokaryotic Kennedy Pathway
TE
LCFACS
Acyl-CoA Acyl-CoA Acyl-CoA
Acyl-ACP Acyl-ACP Acyl-ACP
Free FA
LIPA
SE
LIPA
SE
Remodeling of Membrane Glycerolipids under N-deprivation and High-light Conditions
0
5
10
15
20
25
30
35
40
45
50
DGTS PC PE PI PG MGDG DGDG SQDG
Cont
ent (
µmol
g-1
DW
)
Lipid Class
Extraplastidic Glycerolipids
Nitrogen-Containing
Chloroplast Membrane Glycerolipids
N-replete N-depleted High-light
• Q1: Why did MGDG and DGDG show distinct responses to high-light and N-deprivation stresses?
• Q2: What’s the physiological implication of the dramatic decrease in the PG, particularly under high-light conditions?
• Q3: Is there any high-light or N-deprivation specific
response at the glycerolipid molecular species level?
DGDG (Bilayer Lipid)
MGDG (Nonbilayer Lipid)
Lα Phase HIIPhase
(Laan et al. 2004)
Normal Conditions
Light Harvesting Complex
High-light Conditions Thylakoid Membrane (TM)
Membrane Fusion Mediated by Nonbilayer Lipid
TM
TM
Protein Complex MGDG DGDG PG SQDG PS I Cytoplasm - Cyoplasm - PSII Both Lumen Cytoplasm Cytoplasm
Cytb6f Lumen - - Cytoplasm LHCII - Lumen Cytoplasm -
Location of Lipids within Photosynthetic Complexes (Kern et al., 2010)
PG in PSII (Guskov et al., 2009)
PG in LHCII (Standfuss et al., 2005)
D1
PG in PSI (Kern et al., 2010)
PG1
PG3
PG4
PsaD
PsaE
PsaC
PG3
PG22
PG1
PG2 PG3
High-light response: D1 protein turnover (de novo protein biosynthesis-dependent) N-deprivation response: Down-regulation of PSII/PSI
Protein Complex
Radicals Targets
PS II 1O2, 1Chl, 3Chl, P680+
Lipids, proteins, pigments
PS I O2-, H2O2, OH· Proteins
Photo-oxidative stress occurs when absorbed light energy exceeds the capacity for light energy utilization.
Cellular Content of the Chloroplast Membrane Glycerolipid Species
0
5
10
15
20
25
30
Cont
ent (
µmol
g-1
DW)
N-replete N-depleted High-light
EPA-contaning membrane glycerolipid species are more susceptible to high-light stress.
* *
* *
*
05
101520253035404550
Cont
ent (
μmol
.g-1
DW
)
Lipid Species
N-replete N-depleted High-light
Cellular Content of the TAG Molecular Species
Higher content of TAG species containing EPA was accumulated under high-light conditions.
* * *
*
Conclusions
• Quantatively analysis of 55 molecular species of glycerolipids (covering 9 glycerolipid classes) for the oleagnious microalga N. oceanica IMET1 was achieved by using LC-ESI/MS.
• Lipidome analysis provided insights into TAG biosynthesis in IMET1:
multiple pathways including the de novo Kennedy pathway, PC acyl-editing cycle, and conversion from membrane glyceorolipids contributed to TAG synthesis in this organism.
• Remodeling of membrane glycerolipids occurred under high-light
and N-deprivation conditions, which protected the bilayer thylakoid membranes from fusion and facilitated multiple photoprotection processes.
www.azcati.com Acknowledgements
Sponsored by ASU LightWorks Arizona State University Dr. Gary Dirts Dr. Milton Sommerfeld
Institute of Hyrdobiology, CAS Dr. Qiang Hu Qingdao Institute of Bioenergy and Bioprecess Technology, CAS Ms. Jing Jia; Dr. Jing Li; Dr. Jian Xu