3 ways you can review deconvolution in Progenesis CoMet

In my previous post, I introduced the adduct deconvolution we’ve added to the workflow in Progenesis CoMet and explained how it provides greater confidence in your measurements and identifications and a little of how it works. This time around, I’ll focus on reviewing the deconvolution, with the aim of validating your compounds of interest.

Visualising the results of deconvolution

While the deconvolution process is completely automatic, thereby maximising objectivity, we anticipated that you’d want to review its results too. And Progenesis wouldn’t be Progenesis without high-quality visualisations. 🙂

With that in mind, we created the Review Deconvolution step:

The Review Deconvolution screen in Progenesis CoMet v2.0 
The Review Deconvolution screen, showing an unidentified compound with multiple adduct forms

The Review Deconvolution step helps you to:

  • check that all ions that were grouped as the same compound were grouped correctly
  • find ions that should have been grouped as the same compound, but weren’t

It includes 3 important visualisations:

  • ion map locations: these help you find ions that, perhaps, represent additional adduct forms of the selected compound
  • mass spectra: these help you confirm that all ions grouped in the selected compound have the same neutral mass
  • chromatograms: these help you check that the elution profiles of the selected compound’s ions are consistent (as they should be)

As we’ll see, the mass spectra and chromatograms are especially helpful in understanding why ions weren’t grouped in the selected compound.

1. The ion map locations

In the window shown earlier in this post, the selected compound has ionised with many adduct forms; these are seen most clearly in the series of ion map sections:

Colour-coded adduct forms of a single compound, shown in ion map boxes 
The ion map locations of all adduct forms for our example compound; the same colour-coding of adduct forms is used throughout the software

Each of the boxes here shows a small section of the overall ion map, focussing on the same retention time range, but different m/z ranges. Each m/z range is chosen to show the position of a single adduct form of the selected compound. Within each box, a number of pieces of information are shown:

A small section of the ion map highlighting a single adduct form of the selected compound 
The annotated ion map for a single adduct form of a deconvoluted compound

The ion maps are the primary reviewing tool for false negatives in deconvolution: that is, for finding ions that should have been grouped as the same compound, but weren’t.

In such a case, moving the mouse over a missing adduct’s box shows a small blue circle at the point where the ion’s monoisotopic peak was expected. If, after seeing this, you feel there is a detected ion in the correct location and with the correct charge, you can add it to the compound. To do so, simply right-click on the ion’s outline and select the Add To Compound command.

2. The mass spectrum graphs

The mass spectra of each of the ions in the selected compound are shown in the lower-left panel, with the peak positions for each compound ion transformed into neutral mass values by removing the adduct mass and charge:

The neutral-mass-transformed mass spectrum of a deconvoluted compound 
The graph of mass spectra, showing the perfectly overlaid peaks of correctly deconvoluted ions

The reason for the transformation is that the compound ions’ corresponding isotope peaks should overlay perfectly. If any of the peaks are in positions that aren’t common with the other ions’ peaks, or if the monoisotopic peaks don’t all have the same mass, then it’s clear that the deconvolution is not valid.

Of course, that situation should never arise with automatic deconvolution. However, it’s a very useful indicator of an ion’s validity when you move the mouse over an ion outline in one of the ion map boxes, as seen here:

Clear evidence that the ion in the M+CH3OH+H is not part of the selected compound 
Hovering the mouse over an ion outline shows that its monoisotopic mass is quite different to that of the other adduct forms in the compound

3. The chromatograms

The chromatograms of each of the ions in the selected compound are shown in the lower-right panel of the Review Deconvolution screen. As mentioned in the previous blog post, ions’ retention times and chromatographic profiles must be very similar for them to be grouped as part of the same compound (given that ionisation occurs after the LC stage), resulting in graphs similar to this one:

The scaled chromatographic profiles of a single compound's detected adduct forms 
All adduct forms of this compound have very similar chromatographic profiles, but quite different peak intensities

Notice that each profile’s intensities have been scaled into the same 0-100 range. This allows the shape of the chromatograms to be compared more easily; without this scaling, many low-abundance ions would appear to have a very flat profile and their inclusion in a compound would be difficult to validate. The relative abundances of the different adduct forms are plotted separately in the Peak chart at the right.

As with the mass spectra, this visualisation is especially useful for highlighting ions that don’t belong as part of the selected compound, as it’s easy to distinguish an outlier profile from either the peak’s shape, position, or both:

Clear evidence that the sodiated adduct form of the selected compound is not present in our samples 
Hovering the mouse over the ion in the M+Na ion map shows that its elution profile is quite different from that of the selected compound’s ions.

Further reading

I hope this post has helped to demystify the Review Deconvolution screen and has given you the confidence to review the compounds of interest in your own samples. If there are other details that you would like to learn about – for example, the ion map matrix shown for single-adduct compounds – then the FAQ section on the Review Deconvolution screen is a good place to start.

And finally, if you’ve not yet used the new version of Progenesis CoMet, but are keen to try it out, then click here to download Progenesis CoMet v2.0. I’d love to hear what you think of it. 🙂