Once you have linked a number of spin systems in their correct sequential order you can start to work out which part of the sequence they match up to. In order to do this you need to identify the amino acid type of some of your spin systems. This is harder for double (¹H, ¹⁵N) than for triple (¹H,¹⁵N,¹³C) labelled protein, as the ¹H and ¹⁵N chemical shifts are not as characteristic for their amino acid type as the ¹³C chemical shifts. But based mainly on the ¹H chemical shifts it is nonetheless possible to identify or exclude certain types of amino acids for many spin systems. You can see the distributions of chemical shifts by amino acid type as found in the BMRB within Analysis if you go to Resonance and Reference Chemical Shifts. The following 1H chemical shift table has a visually very appealing way of displaying the average hydrogen chemical shifts. Using this you can quickly see that Alanine, Glycine and Threonine have unusual patterns of side-chain hydrogen chemical shifts. The remainder can be divided into two groups: (1) those with only Hα and Hβ chemical shifts in the 5-0 ppm range (Asn, Asp, Cys, His, Phe, Ser, Trp, Tyr) and (2) those with longer side chains visible in this region (Arg, Gln, Glu, Ile, Leu, Lys, Met, Val). However, always be aware, that not all resonances may be visible in the 15N-TOCSY-HSQC, especially from the ends of long side-chains, and that peaks may be overlapped (especially the two hydrogens from the same CH₂ group often have the same chemical shift).

Once you have identified (or excluded!) the amino acid type of a few of your spin systems in your linked stretch, you can start to compare this to your protein sequence. You may for instance have a stretch which is T-X-A-X-X-Long, where X is, for example, anything other than A, G, T or P and Long is anything with a long aliphatic side-chain. If this motif only appears once in your sequence, then you can make a sequence specific assignment. If you like, you can immediately enter this into Analysis. But I tend to wait until I have assigned most of the sequence and am feeling fairly confident with my assignment before doing this, as it makes undoing mistakes easier – and when doing double resonance backbone assignment it is easier to make mistakes than when you are doing triple resonance assignment! To enter your sequence specific assignment in Analysis, simply bring up the Assignment Panel for one of the peaks in one of the spin systems you want to assign. Select a resonance and click Assign {SpinSystem}ATOM[Resonance]. The Atom Browser panel will appear and you now need to select the exact atom which this resonance corresponds to.

This atom will then be shaded in a slightly darker colour in the Atom Browser panel to indicate that it is assigned. If you have Set Sequential Links between your spin systems, then you should notice that when you assign one of the spin systems, all the sequentially linked ones will become assigned (and shaded in the Atom Browser) at the same time.