The Annotated Proteoform Search node is designed to quickly analyze raw data. This node considers only proteoforms included in your database. This includes any known PTMs, truncations, or amino acid variants that are annotated in the database.

Annotated Proteoform searches can be applied in the following two ways:

  • A narrow tolerance search
  • A wide tolerance search

A narrow tolerance search is usually conducted with a 2.2 Da precursor mass tolerance to account for the common “off-by-one” data deconvolution artifacts. The narrow tolerance search completes quickly and the results do not require further analysis to improve confidence (a few exceptions might include: loss of hydrogen from S-S bonds and deamidation).

A wide tolerance search typically utilizes a 100-1000 Da precursor mass tolerance. This search returns many proteoforms whose precursor mass varies significantly from the theoretical precursor mass. The goal of this search is to find proteoforms that might have an unknown modification resulting in a larger precursor mass error, which could only be found using a wide tolerance. These results require additional work to determine the modification.

Thermo Fisher Scientific recommends opening individual results in the ProSight Lite application to further explore potential modifications. If the modification is identified, it can be added to the database. Then the search must be re-processed to find all proteoforms containing that modification.

To set advanced parameters for an Annotated Proteoforms search, see Advanced parameters of the Annotated Proteoform search.