Table 2.
A General Comparison of PrOF NMR with 1H CPMG as Screening Methods
| 1H CPMG | PrOF NMR |
|---|---|
| Difficult to optimize without known ligand | Does not require reference compound for optimization of experimental conditions |
| More prone to false positives without competition experiments | Less prone to false positives |
| Low concentration of unlabeled protein (2–10 μM) needed | Requires moderate concentration of fluorinated protein (≤40 μM) |
| Low concentration of ligand needed (low to mid μM), solubility of ligands can be observed by resonance height | High concentration of ligand needed (high μM to mM), no information on solubility of ligands is provided |
| No experimental deconvolution needed, but data analysis is time-consuming | Data analysis can be readily automated, but experimental deconvolution required |
| Faster with larger proteins, no theoretical upper bound size limitation | Faster with smaller to medium proteins, approaches a size limitation (<65 kDa for aromatic amino acid labeling)46 |
| NMR time for this 1H CPMG screen (15 kDa protein, three 10 min NMR experiments): 93 h | NMR time for this PrOF NMR screen (15 kDa protein, 2 min NMR experiment): 29 h (23 h of which would be used to deconvolute 140 mixtures; note: only 38 mixtures were deconvoluted to conserve protein) Facile Kd determination with titration experiment |