By initialising the HSQC you select all its peaks and then create new resonances for each peak. In addition, resonances from backbone peaks are grouped into spin systems and their atom types are also assigned.
Begin by peakpicking your HSQC. The easiest way to do this is to zoom out at a fairly high contour level and then drag the mouse over all peaks while holding down Shift and Ctrl. Avoid picking too many artifacts by making sure that only positive peaks can be picked (this option can be selected under Peak Finding in the Peaks menu). You will probably still find that some small positive artifact peaks have been picked which you do not want – delete these by selecting them and pressing Delete. There will also be peaks which have not been picked correctly because of overlap – simply set a peak at the place where you want it by Right Clicking the mouse and selecting Peak and then Add new peak.
Once you are happy that you have picked all the peaks you want, go to Initialise Roots in the Assignment menu. You should make sure that the correct peak list is selected in this pop-up (top left corner). When you click Initialise Peak List! Analysis will assign two resonances to each peak – one in each dimension (resonance numbers are always surrounded by square brackets []). Because Analysis knows that this is an HSQC, it will also create a spin system for each peak, and both resonances from the peak will be added to it (spin system numbers are always surrounded by curly brackets {}). Furthermore, the hydrogen resonances will all be assigned the atom type HN and the nitrogen resonances the atom type N. While this is the correct way to treat the backbone peaks in an HSQC, this is not the correct treatment for side-chain peaks, in particular the Asn and Gln side-chain peaks. Here the atom types are different (Hδ, Hε, Nδ and Nε instead of HN and N) and because we are dealing with NH2 groups, there will be two peaks which belong to the same spin-system and indeed will have the same resonance in the nitrogen dimension. Analysis therefore helps you identify the Asn and Gln side-chain peaks, so as initialise them seperately, and only assign resonances to them, not spin systems and atom types.
The Amide Side Chain Peaks table in the Initialise Roots pop-up lists all the pairs of peaks that Analysis can find which have a nitrogen chemical shift within a certain tolerence of one another. The size of this tolerance can be set in the top right hand box of the Initialise Roots pop-up. When you click on one of the rows in the Amide Side Chain Peaks table, Anaylsis will automatically navigate to that pair of peaks and mark them. The window used for this can be selected from the drop-down menu in the top part of the pop-up.
If a given row has (in your opinion) correctly found an amide side-chain peak pair, then either click Confirm Selected or double-click the cell in the Confirmed? column. This will now switch from No to Yes. Check all the rows. If this has not identified all the amide side-chain peaks, then increase the tolerance (top right hand corner of the pop-up) and click Refresh Table to update it until all pairs are picked up. Once all amide side-chain peaks are confirmed, the initialisation can be done by clicking Initilise Peak List!.
