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Routine Analysis of Five-Microliter Samples using an automated microflow NMR system

Routine Analysis of Five-Microliter Samples using an automated microflow NMR system. Bernhard H. Geierstanger, Ph.D. Protein Sciences Department Genomics Institute of the Novartis Research Foundation 18 Juli 2006 Protasis Web Seminar. 10 - 20 m l. >500 m l.

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Routine Analysis of Five-Microliter Samples using an automated microflow NMR system

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  1. Routine Analysis of Five-Microliter Samples using an automated microflow NMR system Bernhard H. Geierstanger, Ph.D. Protein Sciences Department Genomics Institute of the Novartis Research Foundation 18 Juli 2006 Protasis Web Seminar 10 - 20 ml >500 ml Jansma et al., Anal Chem. 2005 Oct 1;77(19):6509-15

  2. Protasis Micro-coil NMR Probe with CTC-LEAP Sample Changer on Bruker DPX400 • Solenoid microcoil • Active volume: ~2.5 ml • Capillary flow cell: 5 ml • Interface to High Throughput Sample Loader (HTSL pump) • Interface to LEAP autoinjector • Interface to Bruker DPX400

  3. Protasis Micro-coil NMR Probe with CTC-LEAP Sample Changer on Bruker DPX400

  4. Sample Loading Scheme Sample Filter Waste HTSL Pump PAL Injection Valve (6-port) Solvent Reservoir NMR Teflon FEP (4 feet) Filter Sample Loop Stainless steel (5 microliter) Waste Jansma et al., Anal Chem. 2005 Oct 1;77(19):6509-15

  5. Protasis Micro-coil NMR Probe with CTC-LEAP Sample Changer on Bruker DPX400 8mL picked up by syringe 2 8mL injected into 5mL sample loop 3 10mL in well of 384 well plate 1 2.5mL in NMR coil 4

  6. 10 mL in well or vial 8 mL Picked up 5 mL Loaded ~2.5 mL In coil Time for two samples with Pre-load 351s = 5.8 mins 65s 90s 79s 36s 351s = 5.8 mins 65s 30s 90s 79s 12s 36s Samples can be cooled in stacker Next sample can be preloaded Jansma et al., Anal Chem. 2005 Oct 1;77(19):6509-15

  7. Jansma et al., Anal Chem. 2005 Oct 1;77(19):6509-15

  8. Micro-Coil NMR Setup - Modes of Operations Compound 384 well plate 10 mL DMSO 20 mM Library - QC Walk-up Total Recovery Vials 20 mL DMSO 3-20 mM Chemists Serum Samples 384 well plate 10 mL D2O >2 mM

  9. NMR and LC-MS from the same TR Vial 10 mL 3 mM compound (~ 10 mg material) Micro-coil Probe 128 scans 6 mins Jansma et al., Anal Chem. 2005 Oct 1;77(19):6509-15

  10. NMR and LC-MS from the same Total Recovery Vial – Walk-on Applications 20 mM, 32 scans 3 mM, 2 scans, 256 t1 20 minutes C A S/N ~ 54:1 20 mM, 128 scans S/N ~ 223:1 10 mM, 128 scans S/N ~ 97:1 5 mM, 128 scans S/N ~ 61:1 3 mM, 128 scans S/N ~ 46:1 Jansma et al., Anal Chem. 2005 Oct 1;77(19):6509-15

  11. High-Throughput Analysis of Library Compounds from 384 Well Plates C 20 mM compounds in 10 uL deuterated DMSO (left) or protonated DMSO (right) in 384 well plates 128 scans (11 min) with automatic solvent peak identification and suppression 1D NOESY with double presaturation of water (left) (LC1D12) 1D NOESY with shaped pulses and 13C decoupling of water and DMSO (right) (LC1DCWPS) Jansma et al., Anal Chem. 2005 Oct 1;77(19):6509-15

  12. High-Throughput Analysis of Library Compounds from 384 Well Plates C • Throughput ~900 samples per week • Typically ~400 compounds in four 384 well plates • 10ml d6-DMSO per well • spin plate • heat-seal plate • Data processing on instrument • Batch upload to internal database • Analysis by project chemists

  13. High-Throughput Analysis of Library Compounds from 384 Well Plates C • Maintenance tips: • No filters • Stainless steel sample loop • FEP transfer line (100 mm) • Change syringe routinely • Check needle guide • Check injection rotor • Small batches of DMSO • Keep dry • Clean flow probe regularly

  14. High-Throughput Analysis of Library Compounds from 384 Well Plates • Maintenance tips: • Change syringe routinely - every 1500 injection • Check needle guide - when changing syringe • Check injection rotor - for scratches - check when carryover • Small batches of DMSO - 5 ml changed before every weekend run • DMSO in vial with septum and connector for line • Keep dry • Clean flow probe once a month - 45 degree C overnight - 3% hydrogen peroxide/water, flush with water, acid, water, acetone - switch to DMSO • Check shims every month - typically does not change

  15. Glucose Quantification in Mouse Serum 400 Mhz Micro-Coil-Probe (1024 scans) 50 ml serum in 10 ml buffer after lyophilization • Mouse serum • Mouse serum spike with 11 mM glucose • Assignment of metabolites in mouse serum: The spectra are dominated by resonances of amino acids, lactate, creatinine, acetate and glucose PCA analysis of mice on high-fat and normal diet

  16. Glucose Quantification in Mouse Serum 400 Mhz Micro-Coil-Probe (1024 scans) 50 ml serum in 10 mlbuffer after lyophilization 380 mg/dL ~19 mM glucose 54 mg/dL ~2.7 mM glucose Micro-coil NMR setup will be a powerful tool for metabolite analysis especially when combined with separation methods

  17. Acknowledgements Protasis (www.protasis.com) Bob Albrecht Dean Olson Tim Peck LEAP (www.leaptec.com) Wes Moyers NMR Team Tiffany Chuan Ariane Jansma (- 7/2004) Analytical Chemistry Nader Ari John Isbell Director, Protein Sciences Scott Lesley Institute Director Prof. Peter Schultz

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