Release Notes for HX-Express version 2.0
Tested in Excel 2010, 2007, and 2003 within Windows 7 and xp. Not yet tested in Windows 8.
New Features:
- Import delay function to wait for the clipboard to update when pulling in data directly from Masslynx.
The delay will depend on the system performance and architecture, but 350msec seems to work well within Windows 7.
This can be left zero when workng in WinXP.
- Added the option of automatically importing time labels from an open workbook named "Timing" to save time when
importing many sets of spectra.
- Added binomial fitting as an alternative method to centroiding for measuring the mass shift.
This algoryth requires the "solver" add-in within Excel. It also requires the mass envelope of the undeuterated
peptide which can either be read from the undeuterated spectrum, or calculated theoretically from the peptide sequence.
The later requires the Analysis TookPak add-in within Excel. The calculation has been written to handle common
glycan modifications.
- An asymmetric weighting factor can be used for binomial fitting to obtain more accurate fits for overlapped
spectra. This results in larger errors for data points below the theoretical fit, thereby minimizing the
effect of contaminant peaks within a mass envelope.
- Binomial distributions can also be fit againts the raw spectral data. It is much slower, but can be
useful for weaker fragments, where isotope detection is ineffective.
- Double and triple binomial fitting can be applied to any single spectra to deconvolute bimodal/trimodal spectra.
A summary macro is also available for visualizing the results with the aid of a bubble plot.
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Common issues:
Automatic calculation must be enabled within Excel for the routines to function.
In Excel 2003 this is under Tools - Options - Calculations Tab
In Excel 2007+ this is under the main excel window - Excel Options
For importing spectra from masslynx the import delay needs to be used in windows 7. If this delay is too short the data
won't be read and an error will be generated. In rare cases data may still be imported, but it will contain duplicates of
a single spectra. A slight increase in the delay time appears to resolve this issue.
The import spectra function will also reset the CapsLock and NumLock keys.
For binomial fitting the mass window should be truncated right before the monoisotopic peak. If the algorythm finds a
peak with lower M/Z than the monoisotopic peak the binomial distribution fitting won't work.
This provides a more robust measure of the actual extent of exchange because unlike centroids it is not highly sensitive to
changes in peak threshold changes. However since the number of amides used in fitting alters the calculated probability
the same number of amides should be used for fitting all spectra within a series.
Solver needs to be open to activate before HX-Express2 modules will read it in for binomial fitting.
For some systems it will need to be activated every time excel is started before HX-express will recognize it.
For this simply call the solver function (Tools - Solver) and cancel out of it.
For very large peptides (>4000Da) the distribution may become too wide for effective binomial fitting.
In these cases it is best to either make the peak threshold slightly higher to cut down the number of identified
peaks below 30, or use the centroid value. In some cases applying the binomial fit to the raw data instead
of the picked isotopic peaks can be helpful.
In Excel 2007 the prediction of the natural isotopic distribution from the sequence might fail. This is usually because
an earlier version of the analysis toolpak is installed. You can check within visual basic editor whether
the ATPVBAEN has a ".XLA" or ".XLAM" suffix. The ".XLA" is the previous 2003 version.
If many excel worksheets are open the binomial calculations may become sluggish.
The bubble plot for visualizing bimodal data will only plot a single series, therefore if multiple conditions are
processed only the first condition will be plotted.
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For questions or issues regarding the new functionalities contact:
Miklos Guttman
mguttman@uw.edu
University of Washington, Seattle
1959 NE Pacific St. Box 357610
Seattle WA, 98195