Three-dimensional spectroscopy proton resonance assignment

The assignment of resonances to the chemical structure commences from the main chain atoms HN, N, CA, CO and CB. The experiments, i.e. the pulse sequences for this purpose, appear superficially complex but are in fact built from concatenated parts of heteronuclear polarization transfers. The magnetization is often derived from the amide proton and also from the amide nitrogen when using transverse relaxation optimized spectroscopy. Subsequently the polarization is relayed to amide nitrogen and further to carbons. Three-dimensional spectra are produced by acquiring the frequencies of the amide proton directly and recording... 

Correlated spectroscopy (COSY) was among the first two-dimensional (2D) NMR experiment realized [447, 448] and it is still among the most useful NMR experiments. COSY generates cross peaks in the 2D spectrum at the intersection of resonances of coupled spins (Fig. 14.48). In proteins cross peaks are observed for gem-inal, i.e. over two bonds, and vicinal, i.e. over three bonds, protons and in small peptides also couplings over four bonds may be detected. Thus the COSY spectrum allows the identification of spin systems for the assignment. However, apart from peptides, the overlap and degeneracy in chemical shifts is likely to prevent one from obtaining entire spin systems exclusively from the COSY spectrum additional experiments are required. 

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