Semi-automated Assignment

To use the Protein Sequence Assignment function, select this from the Assignment menu and the Protein Sequence Assignment panel will come up. First of all you need to select the window and spectrum from which you do your search, i.e. the CBCANH. Then select the window in which you want the possible matches to be displayed and the spectrum in which you want to find your matches, i.e. the CBCA(CO)NH.

Protein Sequence Assignment Systems pop-up, Settings tab

In the Spin Systems Table tab you will then find a list of all your spin systems at the top, then a list of possible matches for each spin system and in the bottom part of the panel, there are some indictors of the amino acid type and possible sequence position.

Protein Sequence Assignment Systems pop-up, Spin Systems Table tab

When you select a spin system in the top table, Analysis will automatically navigate to this position in your query spectrum (CBCANH) in the window specified previously. It will also immediately look for matches to the peaks in that strip. These are then listed in the Matched Peak Positions box and are displayed as strips in the other window selected previously.

Matching peaks

First of all go to the CBCANH and assign the Cα and Cβ resonances which belong to that strip’s own spin system (i.e. the strong peaks which do not overlap with the CBCA(CO)NH). Simply press a when holding the mouse over each peak, add a New carbon resonance, then Set Same Spin System and Assign Atom Type.

Now you can check whether any of the matches found in the CBCA(CO)NH fit to this resonance. In the example case, spin system {54} is the only one which matches and must therefore be the one which follows {88} in the sequence. In order to assign the carbon dimension of the {54} CBCA(CO)NH peaks and set it as the spin system following {88}, simply select the {54} row in the Matched Peak Positions box and click Set Seq Link.

Making a sequential spin system link

Now you can move on to the next spin system. Simply click Goto i+1 and Analysis will now set spin system {54} as the starting spin system from which to look for matches. It immediately navigates to strip {54} in the CBCANH and shows the CBCA(CO)NH matches. All you have to do is assign the {54} Cα and Cβ, select the match and move on. Simple!

If you cannot find a good match, or there are several that match equally well, just make a note of this and move on to another spin system (e.g. with Goto Next). Note that in the Settings tab, you can experiment with setting your Tolerance to be larger or smaller or with the number of strips to be displayed in the CBCA(CO)NH window.

If you find that you have made a mistake at some point, simply select the spin system in question in the Spin Systems box and click Clear i-1 links, Clear i+1 links or Clear all links and then you can set new links again.