Peakpicking the 3D Spectra

All the peaks in a 3D spectrum should in theory be arranged in columns above the peaks of the 2D root spectrum it derives from (see Visualising 3D spectra for an explanation). Therefore the fastest way of peak-picking a 3D (and also the best way of avoiding picking too much noise) is to pick peaks only in the columns above the peaks you have in your 2D root spectrum. So if, for instance, you are wanting to peak-pick an HNCO or a CBCACONNH spectrum, you would base it on the peaks in your HSQC spectrum. Analysis is then able not only to do the peak picking, but will also assign the nitrogen and hydrogen resonances and atom types from the root HSQC peaks to CBCACONNH peaks.

Before you peakpick each 3D spectrum set your peak picking parameters in the Peaks / Peak Finding pop-up. Make sure that you have selected positive and negative or positive only depending on the peaks that you want to pick in your 3D. Scale relative to contour levels is probably set to 1.0, so you should now set your contour level such, that you only see (and therefore pick) real peaks and not noise. Later you may want to lower the contour level again, but for the main peak picking operation it is usually useful to have the contour level set relatively high.

To peakpick your 3D CBCACONNH spectrum go to the Assignment menu and select Pick & Assign From Roots. At the top of the Navigation Windows tab you need to select your root peak list (i.e. probably your HSQC peak list) and the window in which you want your root spectrum to be displayed. At the bottom of the tab you can select from a drop-down menu the window in which you want to display your 3D spectrum which you are going to peakpick. Now click on Add Window to add this to the list above.

Pick & Assign from Roots pop-up, Navigation Windows tab

Now move to the Spectra & Tolerances tab. This will show all the spectra which are visible in the window you selected in the Navigation Windows tab. Enter suitable tolerances at the top of the tab (something like about 0.02, 0.1 and 0.2 for 1H, 15N and 13C, respectively, should be reasonable starting values). If your 3D contains a water peak or a diagonal (e.g. if picking a NOESY spectrum), then you can exclude these regions using the other tolerance boxes.

Pick & Assign from Roots pop-up, Spectra & Tolerances tab

In the Link Peaks tab you should now get a list of all the spin systems in your root peak list. If you click on one, then Analysis will automatically navigate to its position in the root and 3D spectra in the windows selected previously. When you click on Pick & Assign Root Resonances Analysis will automatically pick the peaks in the 3D and assign the resonances belonging to the root dimension. To start with it may be worth going through some of the spin systems manually in this way in order to check that your tolerances and contour levels seem reasonable. Once you think that you have got these levels about right, you can also get Analysis to peak pick and assign resonances for all spin systems in one go by clicking on Pick All & Assign Root Resonances at the centre bottom of the pop-up. Although this is quicker intially, it can sometimes pay off to go through each spin system individually. Analysis will inevitably make mistakes (see below). You can of course correct these mistakes as you come across them during the assignment process, but often the mistakes can actually slow down your assigning and so you may decide that you would rather go through each spin system one at a time and then be able to start off with a nice tidy set of peaks in your 3D.

Pick & Assign from Roots pop-up, Link Peaks tab

When the peak picking is done you will find that the atom type information as well as the resonance and spin system numbers have been transferred from the HSQC onto the 3D. This means you are only left with having to assign the carbon dimension of the 3D peaks which will speed things up considerably.

Peak picked 3D spectrum

You will almost certainly find that some strips will be stronger than others. Even when you have set all the peak picking parameters as sensibly as you can, you are likely to end up with some strips that contain a large degree of noise and therefore additional peaks have been picked. Simply delete such peaks when you come across them.

3D spectrum with noise

There may be cases where two peaks are very close to one another in the HSQC and within the tolerances you set for the 3D peak picking. In this case you will probably find that that all 3D peaks have been assigned the same resonance and spin system information. If you come across such strips in your 3D, you will need to compare the spectrum carefully with your HSQC and work out which 3D peak belongs to which root peak in the HSQC. Then the assignments on the 3D peak will need to be changed.

Dealing with overlap in a 3D spectrum

In order to do this, you will need to call up the assignment panel. Simply place the mouse over the required peak and press a. This way the assignment panel will immediately show the assignment for that peak.

Assignment pop-up

To change the assignment, in this case from resonances {72}H[143] and {72}N[144] to {155}H[313] and {155}N[314], select the {72}H[143] resonance on the left and click Clear Contrib to remove it from that peak. The new resonance {155}H[313] can be assigned to the peak by clicking on it on the right hand side of the panel. The same process will then have to be repeated for the nitrogen dimension.

Changing an assignment

Once you are happy with the peak picking for each 3D spectrum, save your project and peak pick the next spectrum.