The annotated proteoform search is the standard search mode for top-down proteomics in ProSightPD.
For each precursor mass, a subset of proteoforms within the user-selected mass tolerance are selected from the database. Fragment ions from spectral fragmentation data that has been associated with the precursor mass are then matched against the theoretical fragment ions of the selected database proteoforms. The application determines the number of observed fragment ions matching the fragment tolerance and uses this value to score the identification, as shown in the following figure.
The application calculates scores for each probable MS/MS to candidate proteoform sequence match (PrSM). An observed ion matches a theoretical ion when the two masses are within a user-defined fragment ion mass tolerance. The probability of the observed number of fragment ions matching by chance is then determined and reported as a P-Score.
Although the application queries each proteoform with a theoretical precursor mass in the window, it displays only those proteoforms that meet the user-defined filtering of search results. These filters include the minimum number of matching fragments and false discovery rate (FDR), as shown in the following figure:
The following strategies may be helpful when running annotated proteoform searches:
- Use a large precursor mass window search when identifying an unknown proteoform. If there are several unknown modifications (modifications that are not in the search), a large window search often will still identify the protein. A 100 Da window is a good starting place but a 1000 Da window or larger may be necessary if a very large mass shift is present on the proteoform. Further proteoform interrogation to characterize the proteoform can then be performed once the identity of the protein is known.
- A large number of ions matching one terminus in a protein is evidence of a protein’s identity. You can use the ProSight Lite and the TDValidator Lite applications for further characterization. For more information on these tools, see Use ProSight Lite and Use TDValidator Lite.
- Use Delta M (Δm) Mode to locate unknown modifications near either termini. If the large precursor mass window annotated proteoform search fails to identify a proteoform, consider running another large precursor mass window annotated proteoform search with Delta M Mode enabled.
Activating Delta M Mode increases the likelihood that the search will identify proteins with unknown modifications. However, this mode takes approximately two times longer than the corresponding annotated proteoform search. For more information, see Delta M Mode searches.