A ProSightPD Subsequence search matches MS/MS data against all subsequences of all proteoforms of proteins in a database. The Subsequence search is similar to a bottom-up no enzyme search. The Subsequence search produces many more possible candidates than the annotated proteoform search and will therefore take longer than an annotated proteoform search when using the same precursor window size. The Subsequence search considers every possible subsequence of every base proteoform (unless mentioned otherwise) in the database and attempts to identify any subsequence that matches the observed intact ion mass within a tolerance.
In a typical top-down experiment, not all of the polypeptides identified are intact proteins. A Subsequence search identifies those proteoforms that are a product of biological degradation and cannot be logically predicted. This search compares the observed precursor mass to all possible entries of a particular database within a defined tolerance, for example, less than 10 ppm. The application theoretically fragments those entries that fall within the defined tolerance and compares the observed fragment ions.
NOTE
Use a Subsequence search if an absolute mass analysis fails to identify a proteoform.
A Subsequence search is a two-step process that is repeated for each base protein sequence in the proteome database:
Procedure
- Identify a candidate entry matching an observed precursor mass.
- Calculate all possible theoretical fragment ions for the candidate entry, and then compare the theoretical fragment ion masses to the observed fragment ion masses.
The following figure shows the process involved in a subsequence search.