ProSightPC/PD Software Support – Troubleshooting

There are several possible reasons for this. One common issue the user defined database. Database selection/creation is a critical step in Top-Down data analysis. Foremost the database must include the organisms in the sample or closely related ones. Equally important is the inclusion of PTMs in the database. A protein database made from a FASTA file will not include any PTMs and the result of a search will only return the unmodified protein. In order to screen for modified proteoforms it is recommended that the database be constructed from Uniprot .xml or Uniprot .txt file files, or download from the database warehouse mentioned above (use complex for most comprehensive searches.)

Alternatively, users can reduce the confidence required to return a result. In the event that the MS2 data quality is poor it is likely that any PrSMs achieved are lower scoring and may be filtered out by default.

Lastly ensure that all parameters in the ProSight Software match the data collection parameters (e.g. activation method, resolving power, etc.)

Some modifications may not be included in the PTM annotation in the database and may need to be added manually. Additionally, it is common to find a few abundant proteoforms and several low abundance proteoforms. Low abundance proteoforms often do not yield sufficient fragment ion signal to make a confident PrSM. In this case it may be beneficial to use additional scan averages when fragmenting weak or low abundant proteoforms to improve the fragment ion signal to noise ratio.