> 1. First and foremost, can I MANUALLY follow a peak? (eg "propagate" an
> assignment across the shiftlists?) I get a reasonable grouping with the
> Ax + B fit function but I can't get it to group all the assigned peaks
> in my pH titration. Can I manually add them?
Yes certainly. The grouping will work with any given assignments first,
before attempting a fit.
Incidentally, a buildup curve function for ligand titrations is present in
the new release, but the fitting for the Hill equation isn't yet working
properly, so you'll have to bear with us on that score...
> 2. The dialog asks for a reference peak list.
> Does the corresponding experiment have to be a part of the series
> or can it (or MUST it) be separate?
The reference peak list must correspond to one of the points in the
experiment series, and should carry any known assignments. This is because
you need to have one unquestionable member of each group and be able to
relate at least one member of a peak group with an assignment. This is not
the case for the rate analysis, i.e. you can have an external reference,
because the peaks positions remain static.
> 3. Do I need to specify values in the experimental series to follow peaks?
> For example in my pH titration, does Analysis actually use the pH values
> and uncertainty specified or are they only there as labels?
Yes, it uses the actual values in the series. The peaks in the groups are
found by the closeness of fit of their shift distances, given the
associated sample value, with the fitting function. I also optimises for
straighter 'trajectories'. The uncertainty is not used with the bootstrap
error estimate.
> 4. I think I figured out what some of the options in the dialog mean,
> but what about "Bootstrap errors" and "Min/Max Grad."?
Bootstrap error is an alternative to the sampled error. The bootstrap
error is estimated from the change in fit when random data points are
removed. The alternative is to sample lots of fits within specified error
bounds and to estimate errors based on the width of this distribution.
Min/max grad is just to limit the x/y screen trajectories searched to get
peak groups. This is only really needed to remove ambiguity where there
are few experiments in the series and the shift changes are large.
> 5. I guess the step size in the isotope parameters specifies the maximum
> shift variation between two adjacent spectra in the series, correct?
Yes.
> But then, I'm surprised that peaks which do not vary much at all
> are usually NOT "found" (grouped). Is there a minimum variation to
> be specified somewhere?
This is the shift error in points. The trouble is that when there is
little movement the position error becomes significant and fits can be
poor. The shift error is the amount of 'forgiveness', especially important
if, due to noise, the peak moves in the wrong direction (usually not
allowed).
> 6. Selecting "show fit graph" of a peak group that is not found
> in all spectra of the series causes analysis to lock up without warning.
Fixed.
T.
-------------------------------------------------------------------------------
Dr Tim Stevens Email: [log in to unmask]
Department of Biochemistry [log in to unmask]
University of Cambridge Phone: +44 1223 766018 (office)
80 Tennis Court Road +44 7816 338275 (mobile)
Old Addenbrooke's Site +44 1223 364613 (home)
Cambridge CB2 1GA WWWeb: http://www.bio.cam.ac.uk/~tjs23
United Kingdom http://www.pantonia.co.uk
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