The 3D CBCA(CO)NH experiment is specifically designed to correlate the ¹H and ¹⁵N amide resonances of one residue with both ¹³CA and ¹³CB resonances of its preceding residue via the intervening ¹³CO spin by means of the ¹J(NH), ¹J(N,CO), ¹J(CA,CO) and optional ¹J(CA,CB) coupling constants. A closely related experiment is the 3D HBHA(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  CBCA(CO)NH experiment must be recorded in H₂O.

The original 3D CBCA(CO)NH pulse sequence ( 92JACS6291 ) 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 of duration 1/(4*J(CC)). followed by a A 90º ¹³C pulse to transfer magnetization from ¹³CB to ¹³CA.
3. Fixed evolution delay to achieve antiphase ¹³CA magnetization with respect to ¹³CO via ¹J(CA,CO) followed by  magnetization transfer to ¹³CO by simultaneous 90º ¹³C and ¹³CO pulses.
4. Fixed evolution delay to achieve antiphase ¹³CO magnetization with respect to ¹⁵N via ¹J(N,CO) followed by  magnetization transfer to ¹⁵N by simultaneous 90º ¹⁵N and ¹³CO pulses.
5. ¹⁵N chemical shift evolution during the constant-time t₂ period. Finally, the magnetization is transferred back to the NH protons by a retro-INEPT pulse train and proton acquisition is recorded under ¹⁵N decoupling.

Several improved versions have been proposed incorporating the following modifications:

• Substitution of the first INEPT transfer by a NOE-enhanced version ( 93JB185 and 95JACS4187 ).
• Improved sensitivity incorporating the PEP methodology in gradient-enhanced versions ( 94JMRB203-103 , 95JACS4187 , 93JB185 , and 96JB345 ) and combined with simultaneous 1H and 13C constant-time periods ( 99JMR437-137 ).
• A related 3D [HAHB-CACB](CO)NH experiment has been proposed that uses the so-called projection or reduced-dimensionality technique ( 94JMRB188-105 ). In this experiment, the information of 3D CBCA(CO)NH and 3D HBHA(CO)NH experiments is mixed in a single experiment. Thus, from the F1 dimension of the resulting 3D spectrum, the HB, HA, CB and CA chemical shifts of the preceding residue of a ¹H-¹⁵N amide pair can be extracted.
• Related 3D HN(CO)CACB experiments have been also proposed ( 94JACS11655 ).
• Beta carbon-edited 2D (CBCACO)NH experiment to identify specific amino acids sequencing.
• A similar experiment ( 3D (H)CC(CO)NH experiment) allows to transfer magnetization to all side chain carbon resonances by using and isotropic ¹³C mixing instead of a 90º COSY-type pulse
• A 3D (H)CBCA(CO-TOCSY)NH experiment has been proposed for flexible polypeptides ( 00JB127 ).
• Improved sensitivity and resolution using the TROSY approach ( 99JACS844 ).
• In overlapped spectra it could be advisable to record a slightly modified 3D CBCACO(N)H experiment ( 01JMR246-153 ):
  

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

The CBCA(CO)NH experiment affords a 3D spectrum in which ¹H, ¹⁵N and ¹³CA/¹³CB chemical shifts are displayed in three independent dimensions. Sequential connectivities are due to ¹J(NH) +  ¹J(N,CO) +  ¹J(CA,CO) + optional ¹J(CA,CB).

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