Hi Edward,
answers below:
On 07.01.2013, at 11:43, "Edward d'Auvergne" <edward.dauvergne@xxxxxxxxx>
wrote:
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.
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.
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.
* 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.
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.
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?
Cheers and thanks for the (as usual) quick answer
Martin
Re: Temperature calibration: methanol- vs protein-based,