The 3D CBCANH experiment is specifically designed to correlate the ¹H and ¹⁵N amide resonances with those of the intra- and interresidue ¹³CA and ¹³CB resonances by means of the ¹J(NH), ^1,2J(N,CA) and optional ¹J(CA,CB) coupling constants. The same connectivities can be correlated with an analog 3D HNCACB experiment. On the other hand, inter-residue connectivities can be exclusively extracted from a 3D CBCA(CO)NH experiment.

Implementation on AVANCE spectrometers equipped with a third channel. Improved versions using pulsed field gradients (PFGs) are also available and, therefore, in such cases gradient technology is required.

The experiment is applied on ¹⁵N,¹³C-labeled proteins. Because the amide (NH) protons are involved, the CBCANH experiment must be recorded in H₂O.

The original 3D CBCANH pulse sequence ( 92JMR201-99 ) consisted of the following steps:

1. Initial transfer from ¹H to ¹³C via ¹J(CH) using an INEPT pulse sequence.
2. ¹³C chemical shift evolution during a constant-time t₁ period followed by a a 90º ¹³C pulse to transfer magnetization from ¹³CB to ¹³CA.
3. Fixed evolution period to achieve antiphase ¹³CA magnetization with respect to ¹⁵N and magnetization transfer from ¹³CA to ¹⁵N applying two simultaneously 90º ¹³C and ¹⁵N pulses.
4. ¹⁵N chemical shift evolution during the constant-time t₂ period. Finally Magnetization is transferred back to the NH protons by a retro-INEPT pulse train and proton acquisitionis recorded under ¹⁵N decoupling.

Several improved versions have been proposed incorporating the following modifications:

• Improved sensitivity incorporating the PEP methodology in gradient-enhanced versions ( 96JB315 ).
• Similar experiments using isotropic ¹³C mixing instead of a 90º COSY-type pulse to transfer magnetization from the aliphatic ¹³C to ¹³CA can also be designed (CCANH experiment). In such experiments, all the aliphatic ¹³C chemical shifts of a given amino acid can be correlated with the ¹⁵N-¹H amide pair of the same and the next residue .

  

  

• The CCANH experiment using 1H NOE as a preparation period ((H)CCANH experiment).
  

  

• Sequential 3D CBCANH experiment to obtain only sequential connectivities ( 01JMR328-151 ), similar to the 3D CBCA(CO)NH experiment but avoiding CO magnetization transfer.

More details on practical implementation of the 3D HNCACB experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HNCACB experiment

The HNCACB experiment affords a 3D spectrum in which ¹H, ¹⁵N and ¹³CA/¹³CB chemical shifts are displayed in three independent dimensions. Two different connectivities are present:

• Intra-residue correlations due to ¹J(NH) +  ¹J(N,CA) + optional ¹J(CA,CB).
• Sequential correlations due to ¹J(NH) +  ²J(N,CA) + optional ¹J(CA,CB).

See list of 3D triple-resonance NMR experiments for doubly-labeled proteins.