Total correlation spectroscopy (TOCSY) is a type of two-dimensional nuclear magnetic resonance (NMR) spectrum in which cross peaks result from coherence transfers. For studies of biological macromolecules, TOCSY normally monitors hydrogen atoms (protons), with cross peaks present because of coherence transfer from hydrogen to hydrogen of the macromolecule. TOCSY is a powerful tool for making assignments of proton NMR signals to specific protons of the molecule being examined. The information provided in a TOCSY spectrum contains the data present in a correlation spectroscopy (COSY)-type experiment but includes additional cross peaks that arise because of coherence transfers to or from all protons in a network of spins.
The requirement for the appearance of cross peaks in a proton TOCSY spectrum is the presence of a collection of mutually spin-coupled protons. Consider the alanine residue in the segment of polypeptide shown below:
Protons of the methyl group are J-coupled to the proton attached to the a-carbon (J~7Hz), and the proton on the a-carbon is spin-coupled to the amide N-H proton (J~2-10Hz). The methyl protons are separated from the amide N-H proton by four chemical bonds; thus, the coupling constant between the methyl spins and the peptide N-H is too small to be resolved under typical experimental conditions. Because a resolved coupling constant is necessary for detectable coherence transfer, no such transfer between the methyl protons and the N-H proton would be expected in a standard COSY experiment. A pathway exists for such transfer, however, involving transfer of methyl proton coherence to the a-hydrogen and then transfer from the a-hydrogen to the peptide N-H. The results of these coherence transfers are seen in a TOCSY spectrum as cross peaks that are characterized by two chemical shift coordinates. The coordinate in one dimension corresponds to the precessional frequency of the coherence before transfer, whereas the other chemical shift coordinate corresponds to the chemical shift of the coherence after the coherence transfer process has taken place. In a TOCSY spectrum, an alanine residue will be represented by three diagonal peaks, corresponding to the shifts of the CH3, C aH, and N-H protons, and by six cross peaks, corresponding to all possible origins of coherence and all possible destinations for coherence transfer.
The usefulness of the TOCSY experiment becomes apparent on recognition that the spin-coupling constants are zero between protons on adjacent residues of a polypeptide, so that coherence transfers cannot take place between different amino acid residues of the polypeptide chain. A network of cross peaks in TOCSY can only arise from protons within the same amino acid. If coherence transfer takes place between all possible partners within a residue, the chemical shifts of all spins in that residue can be identified.
Some of the cross peaks present in TOCSY will also be present in a COSY spectrum of the same sample. A significant advantage of TOCSY over COSY is that all components of a TOCSY cross peak tend to be of the same sign, whereas COSY cross peaks have both positive and negative components. COSY cross peaks can therefore become self-canceling when the separation of the cross peak components is inadequate.
The critical element of a TOCSY experiment is the portion known as the isotropic mixing period, during which transfers of coherence take place. The length of this mixing time typically ranges from 20 to 70 ms. For values near the low end of the range, only coherence transfer to nearby spins takes place, and the TOCSY spectrum is similar to a COSY-type spectrum. For longer mixing times, coherence transfers over the entire spin-coupled network are possible. The extent of coherence transfer (reflected in the intensity of a cross peak) is a complex function of the spin-coupling constants in the network, the length of the mixing time, and the method used to achieve isotropic mixing. No assurance exists that an expected cross peak will indeed appear in the TOCSY spectrum for a specific mixing time; thus, for unambiguous identification of all possible coherence transfers in a particular spin system, it may be advantageous to have TOCSY spectra recorded using several mixing times.
The elements of the TOCSY experiment can be built into experiments that produce three-dimensonal or higher NMR spectra. (See also NMR, COSY spectrum, Scalar coupling.)
