ProSightPD 2.0 - FAQs

The E-value is a Bonferroni corrected P Score. Bonferroni correction is a very conservative probability correction factor used to correct for multiple comparisons.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

The P Score is a statistical model of MS/MS matching based on the Poisson Distribution. Learn more and view equations here.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

There are several possible reasons for this. One common issue is 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.)

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Click the Add Experiment icon, or choose Tools > Experiment Adder to open the Experiment Adder dialog box

In the Fragmentation Methods area, select the fragmentation method.

In the Precursor Ion Data/Type box, select the precursor mass type, either Manual or Upload. If you select Manual in the Precursor Ion Data/Type list, select the mass type of the precursor ion in the Mass Type box (either Monoisotopic or Average) and enter the precursor mass. If you select Upload in the Precursor Ion Data/Type list, enter the path and name of the ASCII text file or files containing the precursor ion data in the Text File box, or click Browse to browse for them.

(Optional) Select a predefined search in the box near the bottom of the dialog box.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

Databases with PTMs and FASTA files (no PTMs) can be found here: proteinaceous.net/database-warehouse/

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

RAW files can be read directly into either ProSightPC Software or ProSightPD Software (via Proteome Discoverer). For detailed descriptions of building and executing data analysis workflows in both software, please refer to the ProSightPC and ProSightPD Quick Start Guides.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

For ProSight PC Software:

- Microsoft Windows 7x64 Professional (English version) with current service pack
- ProSight Lite (freeware from prosightlite.northwestern.edu)

For ProSight PD Software:

- ProSightPC
- Proteome Discoverer (x64 version)
- Thermo Proteome Discoverer ThirdParty Components installer (also installs the ProSightPD nodes and ProSightPD with High Mass nodes)
- Microsoft Windows 7x64 Professional (English version) with current service pack
- ProSight Lite (freeware from prosightlite.northwestern.edu)

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.

ProSightPC Software is a standalone software supporting database creation, analysis of infusion data, and high-throughput LC/MS data. ProSightPD Software is a collection of nodes enabling top-down LC/MS data to be searched in the Proteome Discoverer application.

Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.