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

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

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

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

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

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

Lipid purification Image from Triebl et al. Biochimica et Biophysica Acta, 2017 Vol 1862 Courtesy Rachel Morrison

Lipid class purification HPLC Solid Phase Extraction Courtesy Rachel Morrison

Solid Phase Extraction Aminopropyl AP (NH2) Column Pre-conditioned in Hexane Solvent 1- 30 ml Acetonitrile-IPA (2:1) Solvent 2- 10 ml Methanol Solvent 3- 7.5 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) 1 2 3 Courtesy Rachel Morrison

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

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

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

HPLC- Diol column Courtesy Rachel Morrison

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

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

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

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

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

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

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

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

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

Characterisation of extracts by neutron diffraction on D16

Kiesel et al., submitted

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, …

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)?

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