Excluding PSMs with high levels of coisolation - Proteome Discoverer Software - Excluding PSMs with high levels of coisolation - Proteome Discoverer Software - Proteome Discoverer Software

To create a fragment spectrum, you select a precursor mass for isolation, isolate and fragment the ions within a mass window that you define, and record the product ion masses created.

Ideally, you would isolate and fragment only the precursor ions of a single selected component. However, in practice you isolate the precursor ions within a user-specified window—typically 1 or 2 daltons around the isolation mass. Coeluting components with a mass falling into this isolation window are also isolated and fragmented. This process is called coisolation. The coisolating components are likely to be peptides whose fragments are observed in the created fragment spectra. The coisolation can prevent the identification of the selected peptide or lower the identification confidence.

Coisolation is also an issue for reporter ion quantification. In this type of quantification, the peptides from different samples—for example, different treatment states—are modified with isobaric labels. The isobaric labels fragment during precursor ion fragmentation and create reporter tags that appear in the low-mass region of the fragment spectra. The intensity ratio of the observed fragment tags is used for relative quantification of the peptides from the different sample charges.

The coisolating peptides also create reporter tags with the same masses as those from the selected peptide. The intensities of the reporter ions are therefore the sum of the intensities of the reporter ions for all coisolated peptides rather than the target peptide. As a a result, the intensities are perturbed and are not accurate representations of the true abundance of the selected peptide. Furthermore, the perturbed ratios of the selected peptides that are greatly affected by coisolation can also adversely affect the ratios that the application calculates for the proteins that include these peptides.

Determining the extent to which the real reporter tag ratios of the selected peptides are perturbed depends on the level of coisolation and the isolation characteristics of the instrument. The application calculates and displays the percentage of interference within the precursor isolation window. This percentage is the relative amount of ion current within the isolation window that is not attributed to the precursor itself:

The application displays the calculated interference value in the % Isolation Interference column on the PSM and MS/MS Spectrum Info pages. For reporter ion quantification, a high isolation interference value could indicate that a calculated peptide ratio is skewed by the presence of coisolated peptide species.

You can use the Co-Isolation Threshold parameter of the Reporter Ions Quantifier in the Consensus workflow parameter to specify a threshold of between 0 and 100 percent for the allowed coisolation interference. The default value is 100 percent, which means that no PSM is excluded. This parameter is only used for reporter ion quantification, where co-isolated peptides compromise the accuracy of the quantification measurement.