Using LCMS to investigate fatty acid oxidation in cyanobacteria

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Using LCMS to investigate fatty acid oxidation in
cyanobacteria George Taylor
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Cyanobacteria

• Microscopic, unicellular
• Ancient fossils found from 2800 MYA
• Ancestors of chloroplasts in modern plants
• Photosynthetic
• Metabolically diverse

Stahl , 2008
Olson, 2006
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Why are some cyanobacteria interesting from a
biofuels perspective?
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Why is fatty acid oxidation interesting from a
biofuels perspective?
CO2
photosynthesis
respiration
Glycerate-3-phosphate
glycolysis
Acetyl-Coenzyme A
Fatty acid biosynthesis
ß-oxidation
Fatty acids / fatty acyl-ACPs / acyl-CoAs
Acyl-ACP reductase aldehyde decarbonylase
heptadecane
Schirmer et al. 2010
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ß-oxidation

• Major fatty acid degradative pathway

Appears to be lacking in cyanobacteria!
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Hypothesis Cyanobacteria do not have the
ß-oxidation pathway
Testing the hypothesis

• Looking for homology between known ß-oxidation
  enzymes and unknown cyanobacterial protein
  sequences using basic bioinformatics tools
• Detection of the substrates of ß-oxidation
  acyl-CoAs
• Assay of the rate-limiting enzyme of
  ß-oxidation acyl-CoA dehydrogenase
• Metabolite tracing feeding 3H/14C labeled
  fatty acids to cyanobacteria

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Detection of Acyl-CoAs using LCMS-QQQ
Acyl-CoAs are the substrates of ß-oxidation
palmitoyl-CoA (160-CoA)
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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
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Method Development
Standards of palmitoyl-CoA (160-CoA),
palmitoleoyl-CoA (161-CoA) and stearoyl-CoA
(180-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
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MS2 Scans of standards
Precursor masses 161-CoA 1004.5 m/z 160-CoA
1006.5 m/z 180-CoA 1034.5 m/z
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Product Ion Scans
Precursor masses 161-CoA 1004.5 160-CoA 1006.5
180-CoA 1034.5
Fragmentation 135 V
Product ion selection and detection
160-CoA 1006.5 m/z MH
Product ion 499.9 m/z MH
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Product Ion Scans
From this, multiple reaction monitors can be set
up for a range of chain length acyl-CoAs on the
instrument
Product masses 161-CoA 497.4 m/z 160-CoA 499.5
m/z 180-CoA 527.3 m/z
Precursor masses 161-CoA 1004.5 160-CoA 1006.5
180-CoA 1034.5
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Calculating MRMs
Compound Precursor m/z Product m/z
180-CoA 1034.5 527.3
1034.5 28 1062.5 527.3 28 555.3
200-CoA
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-CoAs instrument is set-up to detect
240-CoA 201-CoA 162-CoA 80-CoA
220-CoA 180-CoA 161-CoA 60-CoA
226-CoA 184-CoA methyl-160-CoA 40-CoA
221-CoA 183-CoA 140-CoA propanoyl-CoA
200-CoA 182-CoA ß-hydroxy-140-CoA malonyl-CoA
205-CoA 181-CoA 120-CoA acetyl-CoA
203-CoA 160-CoA 100-CoA
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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
180-CoA 0.293 120-CoA 0.314
181-CoA 0.746 100-CoA 0.25
160-CoA 2.67 80-CoA 0.352
161-CoA 0.584 60-CoA 0.081
162-CoA 0.125 40-CoA 0.903
Me-160-CoA 0.154 acetyl-CoA 2.46
140-CoA 1.03 propionyl-CoA 1.84
ß-OH-140-CoA 0.529 malonyl-CoA 2.29
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Long chain Acyl-CoAs cannot be detected in
cyanobacteria
Acetyl-CoA (0.774 nmol/1x107 cells)
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Method has also been set up to detect and
quantify Carnitines
carnitine
palmitoyl carnitine
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Acknowledgements
Nick Smirnoff Rob Lee Christoph Edner Hannah
Florance Mezzanine Lab Shell Global Solutions
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