1) Partitioning is very unique. Is there a suggested overall approach for applying this Rational Numbers Excel® Add-In to identifying an unknown compound?
It is a completely different approach - made possible by readily available accurate-mass fragmentation data. The Excel Add-In is a very flexible tool, but as a starting point, please see the approach used to extract structural information for chloramphenicol from its accurate-mass fragmentation data. Suggested Approach.
2) What version of Excel is needed to use the Add-In?
The Excel Add-In requires Microsoft Excel 2010, Excel 2013, or Excel 2016 (not included). The Excel must be installed using the default installation which is 32-bit. The operating system required is Windows 7, 8, or 10 (32 or 64-bit). The Excel Add-In will not run on Linux or Mac computers.
The Excel Add-In has a code-signing certificate so that the Excel Add-In can be used even in organizations that have very strict computer security requirements.
3) Does the Excel Add-In work for every compound?
No. There are several factors that affect the results.
First, an assumption is made that there are no major rearangements. For example the base peak of bosentan, with these Excel Add-In partitions, originates from a rearrangement of the molecule in the mass spectrometer. The Excel Add-In results for bosentan puts the migrating methyl group in the wrong position. Less intense fragment ions from rearrangements are usually not a problem.
Second, there is an assumption that the simpler explanations of the mass spectral data are the more likely correct explanations (Occam's razor). In the context of the Excel Add-In, "simpler" means explaining more of the larger fragment ions using as few subfragments as possible. For example, the first (highest scoring) partition of leucine enkephalin, which is Partition 1, has no alignment assigned. This is because there are two glycine moieties but only one subfragment of mass 57 corresponding to glycine. In Partition 1, this problem gives rise to contradictory assignments (and therefore no alignment).
Third, the results of the analysis using the Excel Add-In obviously depends on the quality of the data. More data (e.g. MSn) and better quality data will always improve the outcome. Fortunately, most of the newer Q-TOFs and orbitraps generate excellent data. Contrast the quality of the results for thiamine monophosphate using one actual data set, to the same data modified with the theoretical fragment masses.
You can see dozens of examples of a variety of compounds analyzed with the Excel Add-in here.
4) Can the software be used to interpret the fragments in a spectrum of a known compound?
The software is designed for identifying unknown compounds, but it can be used to assign some fragments. Enter the molecular formula of the known compound as the maximum number of each element so that only that one formula is considered.
5) Is it really necessary to acquire
accurate-mass fragmentation data? Why isn't the accurate mass of
the whole compound sufficient?
This is a very common misconception about
accurate-mass. For example,
xemilofiban has the formula C18H22N4O4.
The PubChem database has 3002 compounds with that same formula
and exact mass and 8469 compounds within 5 mDa of its exact mass (PubChem on 9-11-2014).
In addition, the Excel Add-In makes it much easier and faster to extract structural information from accurate-mass fragmentation data. Perhaps some laboratory groups that depend solely on NMR for identification might be better able to utilize their modern mass spectrometers with this Add-In.
6) The isotope ratio data from my
instrument is sometimes way off. Can the Rational Numbers Excel Add-In work
without isotope data?
The Excel Add-In does require isotope data. The isotope data window ranges from 3% absolute accuracy to 15% absolute accuracy. Few instruments could provide isotope data that would be usable with the 3% accuracy window, because of natural variations in the isotopic abundance of elements and the small ion sample sizes that most modern mass spectrometers use for measurement.