1H-15N HSQC
1H-15N Heteronuclear Single Quantum Coherence Spectroscopy (HSQC)
References:
see Protein NMR Spectroscopy – Principles and Practice by J. Cavanagh, W. Fairbrother, A.G. Palmer III and N.J. Skleton (Academic Press). (Link to Publisher)
Original webpage work by Dr. Vicky Higman (Link to website)
Minimum labeling: 15N (For small peptides/proteins, good results are often obtainable on naturally abunant samples.)
NMR Acquisition Details: 2 dimensions (1H-15N correlated)
Expected Acquisition Time: ~15 mins to 24 hrs (The acquisition of a standard 1H-15N-HSQC experiment is heavily dependent on sample concentration and behavior. However, most cases require ~30 mins to 4 hrs.)
Description: Magnetization is transferred from hydrogen to attached 15N nuclei via J-coupling. The chemical shift is evolved on the nitrogen and the magnetization is then transferred back to the hydrogen for detection.
This is the most standard experiment and shows all H-N correlations. Mainly these are the backbone amide groups, but Trp side-chain Nε-Hε groups and Asn/Gln side-chain Nδ-Hδ2/Nε-Hε2 groups are also visible.
The Arg Nε-Hε peaks are in principle also visible, but because the Nε chemical shift is outside the region usually recorded, the peaks are folded/aliased (this means that they appear as negative peaks and the Nε chemical shift has to be calculated). If working at low pH the Arg Nη-Hη and Lys Nζ-Hζ groups can also be detected, but are also folded/aliased.
The spectrum is like a fingerprint and is usually the first heteronuclear experiment performed on proteins. From it you can assess whether other experiments are likely to work and for instance, whether it is worth carbon labeling the protein. If your protein is reasonably large, you may be able to judge whether deuteration is necessary.
