1. Lipid extraction at the ILL Progress report since Jan 2017
Rachel Morrison, Valérie Laux & Giovanna Fragneto
Soft Matter Science and Support Group
ILL, Grenoble

2. Glass beads & homogenization
Lipid extraction
Freeze-dry & grinding
Cell pretreatment
Glass beads & homogenization
Sonication
Freeze-drying and sonication worked equally – glass beads did not break the cell wall
Courtesy Rachel Morrison

3. Lipid extraction Freeze-dry & grinding Cell pretreatment Sonication
Glass beads & homogenization
Sonication
Beads + Blight Dyer
Sonication + Blight Dyer
Sonication
+ Folch
Standards
Lyso PC PC PS PG PE
Non-polar
Courtesy Rachel Morrison

4. Glass beads & homogenization
Lipid extraction
Freeze-dry & grinding
Denaturation
of lipases
Cell pretreatment
Glass beads & homogenization
Sonication
Courtesy Rachel Morrison

5. Sonication + Blight Dyer
Lipid extraction
Beads + Blight Dyer
Sonication + Blight Dyer
Sonication
+ Folch
Standards
Lyso PC PC PS PG PE
Non-polar
Blight Dyer
Extract
Folch
Extract
No much difference between BD and F so used Blight Dyer – tried soxtec but was giving lots of lyso-phospholipids probably becasue of the heat involved (60 degrees)
Soxtec™
automated extraction
Courtesy Rachel Morrison

6. Lipid extraction Blight Dyer Extract Drying Separated and analyzed
Total lipid extract
Drying
Folch
Extract
Separated and
analyzed
Soxtec™
automated extraction
Courtesy Rachel Morrison

7. Lipid purification
Image from Triebl et al. Biochimica et Biophysica Acta, Vol 1862
Courtesy Rachel Morrison

8. Lipid class purification
HPLC
Solid Phase Extraction
Courtesy Rachel Morrison

9. Solid Phase Extraction
Aminopropyl AP (NH2) Column
Pre-conditioned in Hexane
Solvent ml Acetonitrile-IPA (2:1)
Solvent ml Methanol
Solvent ml IPA-Methanol 3N HCl
First solvent should extract PC, second PE, third PS from literature but we did not get this (tried to change ratio of solvents- no success)
Courtesy Rachel Morrison

10. HPLC- Silica column Operating conditions 0.5 ml/min until 60 mins
Solvents
Hexane 367
Isopropanol 490
25mM phosphate 62
Ethanol 100
Acetic acid 0.6
Courtesy Rachel Morrison

11. HPLC- Silica column Peak 2 Peak 3 Peak 4 Peak 4 Peak 2 Peak 3
Lyso PC PS PE
Non-polar
Peak 2
Peak 3 PC
Peak 4
Peak 4
Peak 2
Peak 3
Courtesy Rachel Morrison

12. HPLC- Diol column Operating conditions Diol column
A: hexane – 2-propanol – butanol – THF – iso-octane – water (64.5:17.5:7:5:5:1) B: 2-propanol – butanol – THF – iso-octane – water (73:7:5:5:10) ammonium acetate was added to both solvents (180 mg/L) Temperature: 65 °C
Composition of solvents from literature, modified to have the gradients
Courtesy Rachel Morrison

13. HPLC- Diol column
Courtesy Rachel Morrison

14. HPLC- Diol column PC Non-polar PS PE Peak 3 Peak 4 Peak 5 PC PS PE
Not reproducible with higher quantities

15. HPLC- Silica column Currently evaluating Si column
Acetonitrile, Methanol, phosphoric acid
Trying less acidic conditions (phosphoric acid degrades the TLC paper and maybe lipids)
Courtesy Rachel Morrison

16. Lipid characterization
Thin layer chromatography,
Gas chromatography,
Ellipsometry,
IR spectroscopy,
Mass spectroscopy,
Light scattering,
Neutron and x-ray scattering.
GC Training probably 8th March
Courtesy Rachel Morrison

17. GC acquired from Shimatsu, to be installed on 7th March in PSCM labs – training 8th March
Courtesy Rachel Morrison

18. Large scale production of deuterated lipids for use by the user communitySi
SiO2
H2O
Neutron experiments
Fermenters for large batches of cell paste
Reconstruction of model membranes
Courtesy Rachel Morrison

19. Future work- Fatty acid incorporation into phospholipids
Yeast grown with
H-Palmitic acid (C16:0)
D-Palmitic acid (C16:0)
H-Oleic acid (C18:1)
Courtesy Rachel Morrison

20. Future work- Fatty acid incorporation into phospholipids
Yeast grown with
H-Palmitic acid (C16:0)
D-Palmitic acid (C16:0)
H-Oleic acid (C18:1)
Reproduced from data in Leitner et al. Biochimica et Biophysica Acta, 2007, Vol 1771
Courtesy Rachel Morrison

21. Future work- Fatty acid incorporation into phospholipids
Yeast grown with
H-Palmitic acid
D-Palmitic acid
H-Oleic acid
Courtesy Rachel Morrison

22. Summary and future work
18 January 2017
Summary and future work
Cell cultures grown in perdeuterated media
Optimisation of lipid extraction protocols
Developing purification methods for lipids
Investigate the effect of fatty acid feed on resulting phospholipid production.
Courtesy Rachel Morrison

23. Characterisation of extracts by neutron diffraction on D16

24. Kiesel et al., submitted

25. Rachel Morrison on leave from 08/17 to 02/18 (contract extended until end of 04/18)
New post-doc hired: Krishna Batchu
To arrive 1st June 2018
PhD in Lipid Biochemistry (Helsinki Institute of Biomedicine)
MSc of Engineering in Bioinformatics (Chalmers)
10 years experience in Lipid Biochemistry/Lipidomics
Experience HPLC, TLC, Mass Spec, Solid Phase Extra(D9) PC species, …

26. Next ILL deliverable 5.9 : Optimization of separation and purification methods due in Sept 2018 (month 36) Can we move it to month 42 (to recover the months of leave of post-doc)?

27. Aknowledgements PSCM at the ILL D-Lab at the ILL Rachel Morrison
SINE2020 post-doc
Robin Delhom
Former PhD Student
Yuri Gerelli
PSCM Coordinator
David Hess
Laboratory Responsible
D-Lab at the ILL
Valerie Laux
D-Lab Research Technician
Michael Haertlein
D-Lab Platform Head