The Subsequence search comprehensively searches subsequences of the isoforms in a specified database. The node considers any PTMs or other annotations found in the database. The Subsequence search is more time- and resource-intensive than the Annotated Proteofrom search. For any given sequence in the database, there could be hundreds of possible subsequences with additional complexity when considering PTMs and other modifications of each subsequence. Due to the increased search space, the Subsequence search utilizes more resources. Use the Subsequence search node to identify unexpected proteoforms in a sample and for discovering protein clipping products and endogenous truncations.
Thermo Fisher Scientific recommends first running the Subsequence search in the most restrictive manner possible and then incrementally increasing the search space as needed. A wide window Subsequence search (>20 ppm) can take days to complete and in cases of very wide precursor windows (>100 Da) may cause computer memory issues. To restrict the Subsequence search space, adjust the following parameters:
- For Precursor Mass Tolerance, set this to 15 ppm or fewer for complex databases.
- For small or targeted searches, tolerances of 2.2 Da can be useful to account for off-by-one errors.