15N-HSQC peak picking

Before any analysis can be made, we need to pick peaks in the spectra that we are going to use. Start by loading the project NapD_2. First, from the 2D 15N-HSQC, we will pick all the peaks that we are confident about that they are not noise peaks. After having created a clean 2D peak list with root resonances, we use these roots to pick peaks in the 3D spectra and propagate assignments from the 2D peaks to the 3D peaks.

2D peak picking

The result of picking the peaks of a NMR spectrum depends on a variety of parameters that can be set in the Peak: Peak Finding popup:

Here we will keep the default values, but select positive peaks only for the 15N-HSQC. The peak picking also depends on the contour level that are set for the spectrum. To avoid picking a lot of noise peaks we start at a high contour base level of ~70. In case the automated peak picking missed some low intensity peaks, we will add them later by lowering the contour level and picking them separately.

In the window HN, press 'CTRL' + 'SHIFT', and draw a box around all peaks:

You should see the peak boxes appear:

Inspecting the automatically picked peaks

The picked peaks should now be inspected, noise peaks removed, and where the routine missed peaks due to overlap we can add new peaks. An example of overlapping peaks is a group of peaks at 8.738/121.307 ppm, for which the peak picker only picked 1 peak:

From the contour levels, one can guess that there are at least 3 peaks overlapping. To verify this, we inspect the CBCAcoNH spectrum, where we expect normally to find 2 carbon peaks per NH, or 1 in case of the presence of a glycine.

To check the number of peaks in the 3D at the NH frequency, click on the peak, and use 'm' to mark the peak. While the peak remains selected, use the right mouse button to navigate to 1H-15N in window HCN (R: Navigate: 1H -15N in HCN):

From the view in the CBCAcoNH it is clear that around the marked line we can identify 3 pairs of CA and CB (i-1) connections at the approximate NH (i) resonances from which we navigated. Picking and marking the CA peaks in the CBCAcoNH, shows where approximately the corresponding peaks in the 15N-HSQC should be located, and manually these peaks can be added:

Likewise, some minor modifications of the peaks can be made in the NH2 region, in the top right part of the 15N-HSQC.

A project with the 15N-HSQC peak list cleaned from noise peaks, and some manually picked peaks added, is saved as NapD_3. This will be the basis for assigning root resonances, and picking the 3D spectra that are used for the assignment of backbone and CB/HB resonances.

Previous: Introduction to protein backbone assignment

Next: Assigning root resonances (1)