Working with variable chemical shifts

Setting up and linking a new shift list

So far during this tutorial all of the chemical shift values we have been recording, by picking peaks and linking resonances & atoms, have gone into one single list. However, there are several circumstances when it is helpful to use more than one list of chemical shifts, so that you may for example separate sets of shifts based upon the experimental conditions they relate to; you may use different shift lists for different temperatures or when doing titration experiments. Commonly in such circumstances the underlying resonances (and hence atoms) that we work with are the same, but the positions of the peaks move significantly and we would not want to average chemical shift values over such a variation.

We are going to set up a new shift list for the second HSQC experiment present in the demonstration project. This second HSQC experiment relates to exactly the same sample (molecule) as the first HSQC but was recorded at a different temperature, which has caused the peak positions to move. Given that the second HSQC experiment is already loaded, all we need to do is go to the M: Experiment: Experiments, {Experiments} and in the |Shift List| column double click on the cell corresponding to HSQC_II. In the pulldown menu that appears simply select "<New>". You will see that a new shift list is now listed. From now on whenever an assignment is made in HSQC_II the shift remains separate from the other spectra. Indeed, any resonances that were already connected to HSQC_II before its shift list was changed will have their shifts recalculated in two separate groups. You could move the experiment back to the original shift list at any time and the chemical shift values will be appropriately recalculated. However, if you disconnect experiments from a shift list (or delete a peak etc) you might have chemical shift values that are not defined by any peaks. Under such circumstances the chemical shifts persist, but are described as "orphans".

This effect can be used to transfer chemical shifts between spectra acquired under different conditions. As an exercise try to open M: Resonance: Resonances, and set ShiftList to 'ShiftList 2'. The table will be empty. Now go back to M: Experiment: Experiments, {Experiments} and change spectrum 'HSQC' to 'ShiftList 2'. The resonance table (M: Resonance: Resonances) now fills up (see figure below), because all the assigned peaks from HSQC feed into ShiftList 2.

Note that the |Shift List Peaks| column says '1' for all shifts; that means that all shifts have one peak in this shiftlist. Now change 'HSQC' back to use ShiftList 1. In the resonance table, |Shift List Peaks| changes to 0:

Theses are the 'orphan' shifts. If you go to window 2 and try to assign a peak, you will notice that the orphan shifts are present and can be used for assignment. To end this exercise go back to the resonance table, click on [Delete Orphans], and say yes when asked if only ShiftList 2 should be used. This removes the orphans again, and leaves us ready for the next exercise.

Propagate assignments to peaks that have moved

Assignment of HSQC_II, with its separately curated shifts proceeds pretty much as assignment normally does, with the only major difference being that for a new shift list (i.e. without any assignments under it) there will be no resonance possibilities that can appear when assigning a peak. - The only known shifts are distinctly different, representing different conditions.

To assign HSQC_II, even though we don't have shifts set, we can say that certain peaks in the two HSQC spectra are equivalent if we can see how they have moved under the different situations. Accordingly we can select a peak in the first HSQC and the corresponding peak in HSQC_II and propagate assignments. That is to say that the peak dimensions link to the same resonances (and hence atoms), even though things have moved. Go to spectrum window1 and try this for the two peaks around 8.33,114.5 ppm (so one peak in each of the HSQC spectra):

Use the mouse with a left click to ensure that these peaks (and only these peaks) are selected. Then in the right mouse menu select R: Assign: Propagate Assignments. Note that this function usually checks to ensure resonance positions are within tolerances, but here with the two shift lists it cannot so the connection is made anyway, which is exactly what we want:

You may like to repeat the above procedure for several HSQC peaks and their counterparts. With a few resonances represented in the new shift list (should be shiftlist 2) have a look at the resonance table (M: Resonance: Resonances) to see evidence of these links. In the resonance table note that you can choose between the different shift lists in a pulldown menu at the top, i.e set the ShiftList: pulldown to "ShiftList 2". Note that when the shift list changes the resonance positions shift and the number of peaks linked also changes. Apart from the resonance table, Analysis also provides direct access to the shift measurements. Looking in the menu at M: Data Analysis: Measurement Lists, you will see that there are tables of the shift values corresponding to each list:

 Note that other types of values like T1 rates, or Hetero-NOE values will also appear as measurements in these tables if they are calculated.

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