Hi, again see below:
>>> One strategy to overcome this was to >>> * record a HSQC/TROSY at different temperatures at the different >>> spectrometers >>> (conveniently by using Brukers multi_zgvt), >> >> Did you use the trosy experiment for relaxation measurements? > > Yes, that's why I'm asking. ;) > > I have the impression that the methanol method is not always perfectly > reflecting the situation in an aqueous buffer with all kinds of salts in it, > and that the huge methanol signals and the resulting radiation damping can > make determination of the "true" MeOH peak maxima pretty difficult. They seem > pretty broad to me. The maximum resolution of peak distance I can get with > Topspin is 0.02 ppm, and if I consider that 0.02 ppm already makes a > difference of ~ 1 K I guess there is the possibility of significant error. We > tried using deuterated methanol at atmospheric pressure (much less intense > signal, sharper peaks, no boiling point artifacts due to odd pressures in a > sealed tube) to see if there are any differences to our standard procedure > with protonated MeOH, but I couldn't see any. For the temperature issues, maybe it is worth talking to the Bruker people. They would know the best. But from my fading memory, I believe MeOH is useful for temperatures < 300 K whereas ethylene glycol is for > 300 K. Your issues might arise from methanol volatility. The different induction and radiation dampening of the standard samples and your NMR sample is an issue, but there is a lot of literature on this that might help you. Again this is ancient memory for me, so you will have to find the articles yourself. There are many different ways for calibrating the temperature. > To make matters worse, we have four solution spectrometers with Bruker > cryoprobes and one usually used with a room temperature probe, and of course > each and avery probe has a different design in the temperature unit. It's not > hard to start believing that every machine is behaving completely differently. Every spectrometer behaves differently with respect to temperature, so temperature calibration is essential for any serious dynamics study. It is incorrect and dangerous to assume that the VT temperature setting is the same as the sample temperature. > To make my point clear I made two figures. > > The following plot shows a series of spectra, which all have the same > reference frequency. You can see that most signals are shifting, into > different "directions". One exception is the (alanine) signal at 134 / 9.2 > ppm, which is pretty stable over a range of 5 K. The series is color coded > red = 305 K to purple = 310 K according to the usual methanol calibration. > > https://dl.dropbox.com/u/4019316/temp-750-zoom.pdf > > Now after adding another spectrum from a different spectrometer (in magenta, > it's also a different sample in this case) I first have the problem of proper > referencing, as I don't know if the diverging signal positions are a result > of slightly inaccurate field calibrations or due to different temperatures. I > referenced it to the said Ala signal which seems to be unimpressed by > temperature changes. > > The "magenta spectrum" should correspond to one of the spectra "in the > middle", namely 307 K, but in reality it fits the 310 K spectrum much, much > better. > > https://dl.dropbox.com/u/4019316/temp-750-reference.pdf > > What do you think? Isn't it odd that the temperature-calibrated spectra don't > fit 100%? They should, and the actual sample seems like a better temperature > indicator to me than a somewhat artificial MeOH sample. > > Currently I'm measuring a whole set of "TROSY-calibrated" spectra, I'll see > if they give me different results in the consistency tests. For permanent reference I have attached the two PDF files you have uploaded to dropbox into a special 'patch' tracker entry at https://gna.org/patch/?3587. The new file links are: https://gna.org/patch/download.php?file_id=16912 https://gna.org/patch/download.php?file_id=16913 >From your spectra (the above file links), it looks suspiciously as though you have an exchange process occurring (though it may not be the case). Have you measured relaxation dispersion data for the system? >>> * select the temperature where the NH spectra are nearly 100% identical >> >> If temperature calibration is important, then you should run the >> experiment on methanol or ethylene glycol to calibrate. Otherwise if >> the spectra are the same but just shifted because of temperature, then >> it's not so important. > > I never saw any differences in MeOH proton peak distance when running the > different T1/T2/HetNOE experiments against a standard methanol sample, > regardless if it was HSQC-based or TROSY-based. Maybe MeOH was not suitable for the temperature used? > If you take your NH spectra from different, MeOH-calibrated > magnets/temperature units and superimpose them – are they completely > identical? Don't you see any differences? I always have to re-adjust my > reference peak lists to find the peaks in the spectra of the different > spectrometers. For me, the spectra are perfectly identical. The only differences are due to spectral noise shifting the peak randomly in height and chemical shift in all directions equally. >> run a quick 1D after running a short version of the experiment. > > That's what I did. I pulsed for 15-20 minutes for the system to equilibrate > and immediatley took a proton 1D to determine the peak distance. > > Maybe I should consider using ethylen glycol since the methanol calibration > method seems to be less suited for temperatures around 300-310 K where we're > working with? Are you determining the distance "by hand" or via a > peak-picking mechanism inside topspin? Do you use deuterated methanol or > standard protonated one? I would recommend ethylene glycol. Have a look at the published literature on NMR temperature calibration and see what is best. Maybe you need to modify the phases on your pulses to preserve the ethylene glycol magnetisation but destroying the water and protein signals, and then use the data from the recorded 2D for your calibrations. This would avoid quick cooling between the experiments. Regards, Edward _______________________________________________ relax (http://www.nmr-relax.com) This is the relax-users mailing list [email protected] To unsubscribe from this list, get a password reminder, or change your subscription options, visit the list information page at https://mail.gna.org/listinfo/relax-users
