To launch the TDValidator Lite application, select TDValidator Lite in the Proteome Discoverer Results view.

The TDValidator Lite application provides a comprehensive overview of the matching fragment ions for a given proteoform and spectrum. The TDValidator Lite window opens with the associated precursor and fragmentation spectra loaded as well as the proteoform from the results table.

NOTE

If the raw files are not in the location specified in the study, TDValidator Lite will not work.

The TDValidator Lite application generates theoretical isotopic distributions based on the exact chemical formula of the proteoform and corresponding fragment ions. These isotopic distributions are then fit to the experimental data. Only ions relevant to the proteoform of interest are considered. As such, the matching fragment ions in TDValidator Lite can be slightly different than those found in ProSight searches from Xtract-detected fragment ions.

In addition to matching terminal ions of a proteoform, the TDValidator Lite application can be used to look at matching internal fragment ions. Because the number of possible internal fragment ions quickly scales for larger proteoforms, several additional features are included in TDValidator Lite to help users better interpret result output. An FDR feature is now included that generates randomized sequences of the same length as the proteoform. A distribution of matches for the randomized sequences can then be produced to understand how many fragment ions, terminal or internal, match on average to a random protein sequence. Another key part of the TDValidator Lite application is the fitter score. In general, scores greater than 0.7 can be considered fairly robust, but scores all the way down to 0.5 can still be acceptable. When looking at internal fragment ions, a higher score can be necessary to limit false positives, particularly for very dense spectral data or large proteoforms (>30 kDa). Lastly, ppm differences between experimental and theoretical ions can be used to limit the scope of the error differences that are allowed. For example, if terminal ions are generally clustering around 0 ppm mass difference but internal fragment ions are much more widely spaced, limiting the allowable mass differences can reduce the false positive internal fragment ions.