CapNMR 'Survey' Probe

Structural Proteomics using NMR Spectroscopy?

Structural elucidation and backbone assignment can now be accomplished with NMR using only a fraction of the mass of protein previously required, significantly reducing the challenges associated with expression.

Using the recently released Protasis/MRM TXI HCN z-Gradient CapNMRTM probe, researchers at The Scripps Research Institute (San Diego, CA) and at Sequoia Sciences (San Diego, CA) have acquired a full compliment of data for complete structural assignment of a protein from microgram amounts.  To demonstrate the detection sensitivity of MicroFlow NMR, these researchers initially evaluated a BPTI sample of 0.24mM concentration on an existing 5uL ICG probe.  A 10 uL injection of sample results in only 2.3 ug of protein residing in the 1.5 uL active volume (original flowcell) of the capillary probe.

Using NMR Spectroscopy as a High Throughput Screening Tool for Folded Proteins!

To further demonstrate the detection sensitivity of the TXI probe, these researchers initially assessed the ability of the probe to serve as a screening tool.  They found that, using only microgram amounts of protein, it is possible to test the folded state of a protein. This tool is extremely vital for testing soluble expressed and purified proteins in the context of structural proteomics initiatives. Only well-behaved and folded proteins are taken forward for structure determination, and therefore the efficiency of the process is dramatically improved if this information can be obtained up-front.  As an example, the 1D 1H NMR spectra of two test proteins TM0979 (well-behaved folded) and TM0731 (poor behavior, not folded) from the hyper thermopile organism Thermotoga maritima are shown.  Well-behaved proteins can be identified by their chemical shift dispersion in the amide and H proton region and by their high field shifted methyl groups, because of ring current shifts from interaction with aromatic groups in the protein core.

Can protein screening really be done effectively at microliter size scales?

For purposes of comparison between conventional and microliter size scales, a series of proteins from Thermotoga maritima were prepared for screening.  Conventional experiments at The Scripps Institute have been carried out in the past using 96 separate proteins from TM.  For microliter scale comparisons, 24 of the 96 TM proteins ranging in size from 10kDa to 40kDa were randomly selected.  The statistics from the conventional experiments were tallied and compared against statistics from the 24 randomly chosen TM proteins.  Examples of the 1H NMR spectra are provided, and the data from the comparison study tabulated below.  These data demonstrate the applicability of capillary-scale NMR as a screening tool for the wider basis set of proteins from Thermotoga maritima.

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