lipidome analysis of the oleaginous...

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)


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)


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)


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)


EPA-contaning membrane glycerolipid species are more susceptible to high-light stress.

* *

* *

*

05

101520253035404550

Cont

ent (

μmol

.g-1

DW

)

Lipid Species


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

lipidome analysis of the oleaginous...

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