Using LCMS to investigate fatty acid oxidation in cyanobacteria

George Taylor

Cyanobacteria

• Microscopic, unicellular

• Ancient – fossils found from ~2800 MYA*

• Ancestors of chloroplasts in modern plants

• Photosynthetic

• Metabolically diverse

*Olson, 2006

Stahl , 2008

Why are some cyanobacteria interesting from a biofuels perspective?

Why is fatty acid oxidation interesting from a biofuels perspective?

CO2

photosynthesisrespiration

Glycerate-3-phosphate

glycolysis

Acetyl-Coenzyme A

Fatty acids / fatty acyl-ACPs / acyl-CoAs

heptadecane

Acyl-ACP reductase + aldehyde decarbonylase*

Fatty acid biosynthesisβ-oxidation

*Schirmer et al. 2010

β-oxidation•Major fatty acid degradative pathway

Appears to be lacking in cyanobacteria!

Hypothesis – Cyanobacteria do not have the β-oxidation pathway

Testing the hypothesis:

• Looking for homology between known β-oxidation enzymes and unknown cyanobacterial protein sequences using basic bioinformatics tools

• Detection of the substrates of β-oxidation; acyl-CoAs

• Assay of the rate-limiting enzyme of β-oxidation; acyl-CoA dehydrogenase

• Metabolite tracing – feeding 3H/14C labeled fatty acids to cyanobacteria

Detection of Acyl-CoAs using LCMS-QQQ

Acyl-CoAs are the substrates of β-oxidation

palmitoyl-CoA (16:0-CoA)

Extraction and sample preparation

3 cyanobacterial strains and E. coli (positive control) were harvested at an OD of 4 by centrifugation, homogenised and extracted in acetonitirile/isopropanol/KH2PO4 at pH 6.7

Acyl-CoAs are acidified and enriched by SPE using a 2-(2-pyridyl)ethyl silica gel column, eluted at pH 7, dried and resuspended in water

Minkler et al. 1999

Method Development

Standards of palmitoyl-CoA (16:0-CoA), palmitoleoyl-CoA (16:1-CoA) and stearoyl-CoA (18:0-CoA) were used at a concentration of 75 μM in water

HPLC:Acyl-CoAs eluted isocratically on a 30 mm x 2mm reverse phase column (3.5 μm particle size). Mobile phase is 55% ACN and 45% 10 mM ammonium acetate in water. Run time 3 min. Wash and re-equilibration time 7 min to eliminate carryover contamination

MS-QQQ:Acyl-CoAs are ionised by ESI (positive polarity).

Veld et al. 2009

MS2 Scans of standards

Precursor masses:

16:1-CoA 1004.5 m/z16:0-CoA 1006.5 m/z18:0-CoA 1034.5 m/z

Product Ion Scans

Q1 Collision cell (Q2) Q3

DetectorIon source

Precursor masses:16:1-CoA 1004.516:0-CoA 1006.5 18:0-CoA 1034.5

Fragmentation:135 V

Product ion selection and detection

16:0-CoA 1006.5 m/z M+H

Product ion = 499.9 m/z M+H

Product Ion Scans

Product masses:16:1-CoA 497.4 m/z16:0-CoA 499.5 m/z18:0-CoA 527.3 m/z

Precursor masses:16:1-CoA 1004.516:0-CoA 1006.5 18:0-CoA 1034.5

From this, multiple reaction monitors can be set up for a range of chain length acyl-CoAs on the instrument

Calculating MRMs

Compound Precursor m/z Product m/z

18:0-CoA 1034.5 527.3

1034.5 + 28 = 1062.5

527.3 + 28 = 555.3

20:0-CoA

-CoAs instrument is set-up to detect

24:0-CoA 20:1-CoA 16:2-CoA 8:0-CoA

22:0-CoA 18:0-CoA 16:1-CoA 6:0-CoA

22:6-CoA 18:4-CoA methyl-16:0-CoA 4:0-CoA

22:1-CoA 18:3-CoA 14:0-CoA propanoyl-CoA

20:0-CoA 18:2-CoA β-hydroxy-14:0-CoA malonyl-CoA

20:5-CoA 18:1-CoA 12:0-CoA acetyl-CoA

20:3-CoA 16:0-CoA 10:0-CoA

A wide range of –CoAs are detected in extracts of E. coli

Compound nmol acyl-CoA / 1 x 107 cells

Compound nmol acyl-CoA / 1 x 107 cells

18:0-CoA 0.293 12:0-CoA 0.314

18:1-CoA 0.746 10:0-CoA 0.25

16:0-CoA 2.67 8:0-CoA 0.352

16:1-CoA 0.584 6:0-CoA 0.081

16:2-CoA 0.125 4:0-CoA 0.903

Me-16:0-CoA 0.154 acetyl-CoA 2.46

14:0-CoA 1.03 propionyl-CoA 1.84

β-OH-14:0-CoA

0.529 malonyl-CoA 2.29

Long chain Acyl-CoAs cannot be detected in cyanobacteria

Acetyl-CoA (0.774 nmol/1x107 cells)

Method has also been set up to detect and quantify Carnitines

carnitine palmitoyl carnitine

Acknowledgements

Nick SmirnoffRob LeeChristoph EdnerHannah FloranceMezzanine LabShell Global Solutions