Post on 20-Jan-2016
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