Generating a report for a gel-based project

This is a step by step guide on how to generate a report of protein identifications with gel spot IDs using Proteios. The Hits report can be found under the active project by selecting <Active Project> -> Reports -> Hits.

For the process to be as automatic as possible, it is important to name peak list files in such a way that the file name before the file extension ends with underscore ('_') followed by the plate position ('A1'). A good file name would be: FF_070603_PLATE1_A12.RAW, where FF are the initials of the experimenter, which is followed by the date. The plate Id is PLATE1, and the position of the sample in the plate was A12.

Uploading files

The files you will need to upload are:

1 The output files from your spot handler. From the Spot Handling Workstation these are xml files found in the log.
2 The processed peak lists from the mass spectrometer. For MS/MS files that are not in mzData or MGF format it is advisable to convert the files, which can be done in Proteios (see below)
3 Search results from search engines. Preferably the searches should be conducted with mzData or MGF files as input

It is a good idea to place these files in separate directories. If you have peak list files that come from analysis of different microtitre plates or MALDI targets, they should be placed in one sub-directory per plate.

In the special case of Piums searches, you should upload the entire folder with the *_pepfil.xml and *_matcher.xml files. No file conversion is needed.

Peak List conversion

The preferred peak list format is mzData. It is possible to create reports also if searches are done with MGF or PKL files, but the generation of combined hits reports on the peptide level will fail if mzData or MGF were not used for the searches.

To convert files first upload them to you Proteios server. Browse to the directory with the files and select all the files you want to convert by checking the select boxes. Then choose Extensions->Convert file[s] and select the appropriate file conversion plugin. Then click the 'next' button. The conversion jobs will be generated. The output files will be located in the same folder as the input files, and you can download them via ftp and then perform the searches.

If you have a Protein Lynx Global Server project where you processed the files, it is best to convert the xml peak lists that can be found in that project, and not the PKL files, since retention time and processing information is lost in the PKL files. There is a Perl script that will rename the PLGS files and place them in one folder for you.

ProteinLynx Global Server to mzData/mzML conversion

The plugin to convert QTOF-MSMS files from PLGS to mzData is the ProteinLynx Global Server to mzData converter. It will by default state that the instrument used was an QTOF Ultima. If that is not true, you need to place a file named 'PLGSInstrument.xml' in the same folder as the files to be converted. It should contain the xml of the instrument section of mzData as in this attached file

Importing the files

The easiest way to import the data is to use the "Hits import wizard' that can be found under the Hits report. If you have a system with separate spot picker and digester, the first step of the wizard can currently not be used. The ExQuest instructions will have to be followed instead.

1. The first step is to import the gel spot to microtitre well or MALDI target position from the spot picker log file. You do not have to enter the Gel ID, but if this is done, only the mapping for that particular gel will be imported. Then click 'next' and select the robot result file (Spot picker log file). You will then be transferred to the job queue view, and here you can see when the import is ready.

2. When you go back to the Hits Report again, you should see that it is now populated with gel spot numbers and plate information. It is then time to register the peak list files. This is done in the Hits import wizard for one plate at the time. Select the plate id and then the peak list files that were used for the protein identification searches. Usually this would be the mzData files. For Piums searches you should select the _pepfil.xml files, but you can select all files in the directory, and the results will be the same apart from warnings that _matcher.xml and _pepex.xml file are not imported.

3. The final thing to import are the search results (step 3 in the wizard). You can import all search results in one go, provided that the input peak lists have been registered (step 2) and that they are located in the same directory.

Using the report

You can navigate in the report and click on some fields to get more information. Filtering of the report is performed by entering filter expressions in the filter fields for columns supporting filters. It is also possible to export the entire report as a tab-separated file by clicking 'Export to File'. The output file can be downloaded and open up as a spreadsheet in programs like Star Office - Calc or Microsoft Excel.

Combined hits report

If searches for gel spots have been performed with more than one search engine, you can use the Combined Hits Report to create a report which will combine the search results and allow you to make a report with a predefined error rate. The report will be written to a tab-separated file that you can download. In the current version, search results from Mascot, X!Tandem and Piums are considered in the report. For details about how the report is generated, see  this article. X!Tandem results from searches with different pluggable scoring algorithms are treated as separate search engine searches.

There are some parameters for the report:
The gel Id is the external Id of the Gel, and it should be written exactly as it is written in the Hits report.
The maximum FDR is the maximum false discovery rate that is tolerated. For smaller dataset this may have to be set higher than the default for any results to pass.
If compare peptides is checked, the combination of search results is done on the peptide level rather than on the protein level. For 2D gel projects this is usually not that good, but for 1D gel or MudPIT combinations the comparison should be on the peptide level.
The random hits prefix is the prefix for entries in the random or reverse part of the database.
Output file name is the name of the report file that is written. If the file exists, a count will be added to the end of the ifle name, so no file will be overwritten.