The 3D H(CA)NH (or H(CA)NNH) experiment is specifically designed to correlate ¹⁵N and NH chemical shifts with the intra- and interresidue ¹HA carbon shifts via the intervening ¹³CA spin by means of the ¹J(NH), ^1,2J(N,CA) and ¹J(CH) coupling constants.

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 H(CA)NH experiment must be recorded in H₂O.

The original 3D H(CA)NH pulse sequence ( 91JMR84-91 ) was based in an analog 2D sequence ( 90JMR183-87 ) and consisted of the following steps:

1. After the initial 90º ¹H pulse, ¹H chemical shift evolution during the variable evolution t₁ period takes place.
2. Fixed evolution delay to achieve antiphase ¹H magnetization with respect to ¹³CA via ¹J(CH).
3. Magnetization transfer to ¹³CA by applying simultaneous 90º ¹H and ¹³CA pulses.
4. Fixed evolution delay to achieve antiphase ¹³CA magnetization with respect to ¹⁵N via J(N,CA).
5. Magnetization transfer to ¹⁵N by applying simultaneous 90º ¹⁵N and ¹³CA pulses.
6. ¹⁵N chemical shift evolution during the variable evolution t₂ period followed by evolution of antiphase ¹⁵N magnetization with respect to ¹H.
7. Back transfer to the NH protons by a retro-INEPT pulse train.
8. Proton acquisition under ¹⁵N decoupling.

Several improved versions have been proposed incorporating the following modifications:

• The same involved backbone nuclei can be used in analogs 3D (HA)CANH and 3D HACA(N)H ( 95JMRA19-113 ) experiments:
    Related experiments are 3D HA[CAN]NH ( 93JACS9307 , 93JB127 , 94JMRB294-104 , 95JMRB89-108 ) 3D HXYH experiments.
• A related experiment has been proposed (3D HN(CA)H or HN(CA)HA experiment) in which the transfer mechanism has been reversed ( 92JMR406-100 and 92JB203 ). In the same way, some variants have been proposed: 3D H(N)CAHA ( 92JB203 ) 3D (H)NCAHA ( 92JB25 ), 4D HNCAHA , and 3D HNN[CAHA] ( 02JB41-24 ), and multiple-quantum 3D trosy-HN(CA)HA and 4D trosy-HNCAHA ( 03JB69-26 ) experiments .

The H(CA)NH experiment can be recorded in automation mode.

More details on practical implementation of the 3D H(CA)NH experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D H(CA)NH experiment

The H(CA)NH experiment affords a 3D spectrum in which ¹H, ¹⁵N and NH chemical shifts are displayed in three independent dimensions. Two different peaks can be present:

• Intraresidue connectivities due to ¹J(CH) +  ¹J(N,CA) + ¹J(NH)
• Weaker interresidue connectivities due to ¹J(CH) +  ²J(N,CA) + ¹J(NH)

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