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  l >500  l Jansma et al., Anal Chem Oct 1;77(19):

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

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

Protasis Micro-coil NMR Probe with CTC-LEAP Sample Changer on Bruker DPX  L in well of 384 well plate 2 8  L picked up by syringe 3 8  L injected into 5  L sample loop  L in NMR coil

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

Micro-Coil NMR Setup - Modes of Operations Compound 384 well plate10  L DMSO20 mM Library - QC Walk-up Total Recovery Vials20  L DMSO3-20 mM Chemists Serum Samples 384 well plate10  LD 2 O>2 mM

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

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

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 13 C decoupling of water and DMSO (right) (LC1DCWPS) Jansma et al., Anal Chem Oct 1;77(19):

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

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

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 High-Throughput Analysis of Library Compounds from 384 Well Plates

Glucose Quantification in Mouse Serum 400 Mhz Micro-Coil-Probe (1024 scans) 50  l serum in 10  l buffer after lyophilization A)Mouse serum B)Mouse serum spike with 11 mM glucose C)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

Glucose Quantification in Mouse Serum 400 Mhz Micro-Coil-Probe (1024 scans) 50  l serum in 10  l buffer 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

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