This article is part of a troubleshooting path. If the symptoms below do not precisely match your issue, or if the solution has not fixed the issue, consider starting at the beginning of the path using this link
OBJECTIVE or GOAL
Maximize the number of proteins identified from MSe or HD-MSe data by optimizing the PLGS spectrum processing parameters.
- ProteinLynx Global Server (PLGS)
- Progenesis QI for Proteomics v3
- MSe, HD-MSe, or SONAR data
- PLGS Threshold Inspector (a tool for automating optimization of PLGS MSe processing parameters)
- PLGS (Tesla GPU equipped) processing PC
NOTE: Progenesis QI for Proteomics v4 includes an automated MSe threshold optimization routine, and does not require PLGS Threshold Inspector.
To use PLGS Threshold Inspector to optimize MSe processing parameters, you need the following:
A typical MSe RAW data file from the dataset you want to analyze
PLGS (you need to use the Apex3D64.exe, Peptide 3D.exe, and iaDBS.exe files) or Progenesis QI for Proteomics v3
A PLGS search workflow exported as an XML file
A sequence database (fasta file) appropriate for the sample used to generate the raw data and referenced by the workflow file
Detailed instructions for setting up and using PLGS Threshold Inspector, with screenshots, can be found on UC.
Download and install PLGS Threshold Inspector from http://sourceforge.net/projects/plgs...urce=directory.
Export a search workflow appropriate for the sample to be analyzed from the PLGS workflow library as an XML file (for instructions, see the PLGS help files).
Open PLGS Threshold Inspector.
At the top-left of the PLGS Threshold Inspector window, select the raw data file you want to use for optimization.
At the top-right of the window, you may see a flashing red icon. Click on the icon next to that and it will open a drop-down menu used to link to the required EXE files. The example paths below are for PLGS3.0.3 and may differ for other versions of PLGS.
Apex3D - C:\PLGS3.0.3\lib\apex3d\ (select either Apex3D.exe or Apex3D64.exe)
Peptide3D - C:\PLGS3.0.3\lib\apex3d\Peptide3D.exe
IADBs - C:\PLGS3.0.3\bin\IADBs.exe
Workflow - Select the workflow XML file containing the search parameters exported at step 2
Select the fasta file containing the sequence library referenced by the workflow XML file exported at step 2
NOTE: Progenesis QIp v3 users also have PLGS3.0.3. Both use the same executables for MSe processing. So Progenesis QIp v3 users can either follow the above instructions or use the executables from the Progenesis QIp installation folder, as described in the attached document.
- Select the time window in your data that you want to test the parameters on.
Suggestion: When you first set up PLGS Threshold Inspector, select one set of parameters and a narrow time window to ensure that all links are working.
- Select the parameter combinations that you want to test by selecting the check boxes on the left-hand side of the main window.
- Press Play and leave it to work through the parameters and find the most appropriate ones for the data file you have selected.
Note: PLGS Threshold Inspector reports the number of proteins, not the number of hits. This means that the results may appear inflated due to inclusion of redundant homologues.
- Create MSe Processing Parameters in PLGS based on the results of the PLGS Threshold Inspector comparison.
- Neither the PLGS or Progenesis QI Proteomics default MSe processing parameters, nor the parameters listed in the PLGS system suitability test document are universally applicable to all samples.
- Setting MSe processing parameters incorrectly can have a dramatic adverse affect on the number of proteins identified.
- Setting MSe parameters low to be on the safe side can actually result in fewer hits as PLGS penalizes unmatched peaks.
- Users are strongly advised to optimize Low Energy and High Energy threshold settings for each of their sample types.
- PLGS Threshold Inspector is a tool for automatically finding the optimal LE and HE thresholds for PLGS data processing.
- PLGS Threshold Inspector was developed at Waters, but it is not official Waters software and is distributed as freeware.