Pick and assign 3D peaks from 2D root peaks

Instructions

3D peak picking

Peaks in a 3D or higher dimensional spectrum can be picked in the same way as in 2D, by picking the peaks in the 2D planes of the higher dimensional spectrum. When picking peaks in a 2D plane of a higher dimensional spectrum, one only has to take into account that the visible peaks may have their maxima in neighboring planes and therefore may not be picked immediately. To pick peaks that have their maxima in neighboring planes, you can either:

• • navigate in the orthogonal dimension and find the maximum of the peak that you want to pick 

  • set the parameter "Extra thickness in orthogonal dimensions" in the Peak: Peak Finding popup to a higher value.

3D peak picking from 2D root peaks

In case we have for example a 15N-HSQC spectrum with its peaks picked, we can use these peaks to more efficiently pick 3D peaks in 3D spectra that have the same 1H and 15N dimensions. The procedure works by searching peaks in the 3D spectra only around the HN root peaks. For example, a 3D CBCA(co)NH spectrum can be picked efficiently using the 15N-HSQC peaks, since these spectra have the 1H and 15N dimensions in common.

A manual procedure to pick the 3D peaks from 2D root peaks could be as follows:

• • In the 2D root window (e.g. an HN window), select a peak and for convenience mark it with 'm'.

• From the peak center use R: Navigate: 1H - 15N in "Target window" to navigate to the 3D window.

  • In the 3D window, pick the peaks around the marked frequency using a normal 2D peak picking procedure.

Example

To illustrate how to find and pick peaks in a 3D window, we use a 2D 15N-HSQC spectrum (displayed in an HN window) in addition to a 3D CBCA(co)NH spectrum (displayed in an HCN window). Now we first navigate from the 2D spectrum to the 3D spectrum using the 1H and 15N frequencies of a peak of interest in the 2D spectrum using R: Navigate: 1H - 15N in HCN:

The CBCA(co)NH spectrum is now shown at the corresponding H and N positions that were used to navigate from the 15N-HSQC:

Now we pick the 2 peaks in the CBCA(co)NH as if it was a 2D spectrum, by holding down 'CTRL' and 'SHIFT' while drawing a rectangular box with the left mouse button:

The two picked peaks that lie at the 1H vertical marker line can be assigned to the same root resonances as the 2D peak from which we started navigating. The wrongly picked peak can easily be removed by marking it and clicking 'del'.

Semi automatic 3D peak picking

Instead of picking all the peaks manually in 3D spectra as described above, there is a semi automatic procedure in Analysis that uses the peaks from the 2D spectrum to search the 3D spectra around the root frequencies for peaks, which it then can pick and assign. This procedure avoids picking of noise peaks, and at the same time provides a convenient way to perform the necessary manual inspection of peaks and their assignment. The peaks picked in the 3D spectra will in the example below be assigned to their root resonances, it is however possible to expand this semi automatic procedure to also deal with peak assignment in the non-root dimension.

The semi automatic peak picking procedure is accessible from M: Assignment: Pick & Assign From Roots. This raises Assignment: Pick & Assign From Roots. This popup has three tabs:

• {Windows and Spectra}. This tab has two tables 'Target Windows' and 'Assignable spectra'. The 'Target Windows' specifies which 3D windows will be used, and the user has the option to set parameters for the associated spectra in 'Assignable Spectra'. The spectra that are made active in the "Active" column will be used for picking and assignment in the root dimensions. The "Assign Non-root dim?" column specifies wherever the user might want to assign the non-root dimension as well. Make sure that the correct root window is chosen in the upper right part of the tab.

• {Tolerances}. The tolerances for peak finding based on root frequencies can be modified in this tab if desired. 

• {Link Peaks}. In the last tab, the semi automatic peak picking and assignment procedure is actually performed. Each row in the tab corresponds to a root peak and clicking on a row allows the user to pick and assign 3D peaks in the target spectra around the root frequencies, using the button [Pick & Assign Root Resonances]. To inspect the peaks that have been picked, the user can manually navigate to the correct position in the 3D, but the most convenient way is to check the "Navigate to root" and "Navigate to targets" options. This will automatically move the spectrum views to the positions where peaks can be picked and assigned when selecting a root peak row. After picking peaks, the peak assignments can be inspected by clicking 'a' or R: Assign:.. on a selected peak. This raises Assignment: Assignment Panel in which the root dimensions now should have been assigned based on resonances, spin systems and atoms from the root peak. After that the picked peaks have been inspected, click [Next Row] or on a optional row to pick and assign peaks further. 

• Note: it is possible to use the option [Pick All & Assign Root Resonances] with appropriately set tolerances for picking and assigning the complete spectrum at once. This procedure works well for non overlapping peaks. However, overlapping peaks still need to be investigated manually, and sometimes peak positions have to be changed or manually picked. To avoid problems later on, we advice to either go through the complete list of roots one by one using [Pick & Assign Root Resonances], check that all peaks are correctly picked and assigned and modify the peaks and assignments where needed. Alternatively, pick and assign all peaks at once, and scan through the peaks later to remove unwanted peaks and add peaks where the peak picker failed. A well curated peak list will speed up procedures later on.

Example

To demonstrate the semi automatic peak picking procedure, we will pick and assign 3D peaks in CBCA(co)NH, HNCACB spectra in a HCN window, and HN(ca)HA and HBHA(cbcaco)NH spectra in a HHN window, from 2D 15N-HSQC root peaks.

In this example, all the 2D root peaks have been picked and assigned in the HN window and we start by accessing Assignment: Pick & Assign From Roots through M: Assignment: Pick & Assign From Roots. In {Windows & Spectra}, we ensure that 'HN' is selected in the "Root Window" pulldown menu. Then we make the 'HCN' and 'HHN' windows available in 'Target Windows' by selecting them one by one in the pulldown menu and clicking [Add Target Window:]. We make sure that in "Assignable Spectra" all spectra are active, and in this case we do not assign the non-root dimensions, nor do we use the 15N-NOESY-HSQC:

Now we set the parameters in the {Tolerances}:

and go on to {Link Peaks}, where we select a row in the table for a root peak to start from:

Because "Navigate to root" and "Navigate to targets" were checked, the selection of the row has prompted the HN window to center on and mark the selected root peak:

and the HCN and HHN windows have navigated to- and centered on the HN coordinates of the 2D peak. Next, we click [Pick & Assign Root Resonances], the result is that for all the 3D spectra, peaks have been picked and assigned to the same root frequencies.

We toggle the spectra on and off and navigate +- 1 plane in the Nitrogen dimension to verify that all peaks have been picked properly, and no unwanted (noise) peaks remain. This procedure needs to be repeated for all other NH root peaks by clicking on the rows in {Link Peaks}, or using the [Next root] and [Previous Root] buttons.