2D Gel-based proteomics workflow
This is based on the workflows at: http://www.proteomics.swegene.lu.se/
Proteios should provide support for sample-tracking for a gel-based workflow. the workflow is divided into a number of steps. During each lab step files will be generated that have to be imported into Proteios. Proteios should be able to keep track of the experiments and provide sample tracking. At the end of the workflow it should be possible to generate reports.
The first step is to run a number of 2D-gels for different samples. On the gels proteins are separated and are visualised as spots. The gels are scanned and the gel pictures are then matched and compared using the Decyder or ImageMaster software packages. Proteios should at a later stage be able to import files from these programs and perform statistical analyses on the differences in spots between samples.
When interesting spots have been found preparative 2D-Gels are run and matched against the analytical gels. A pick list is generated and exported to the Spot Handling Workstation (SHW). The SHW will treat the spots so that the proteins can be identifed.
In the SHW spots are excised from the gels and transferred to 96-well microtitre plates (MPs). In the MPs the spots are digested with Trypsin to generate peptides that are extracted and transferred to another MP. Depending on which type of mass spectrometer (MS) will be used to analyse the samples they can be further spotted onto a MALDI target plate or retained in the MP. The SHW will return an XML file which describes how the samples where processed, and most importantly which spots end up where. To start with Proteios has to be able to read these files and keep track of which spot is placed in which MP and at which position. The schema for the files is in Proteios1 called 'spot.xsd'. In Proteios1 the tracking information is found in the Spot elements as MPId,MPLabel,target_id and target_position. Ideally information about the processing should also be saved for the gel, but this is not an urgent feature.
The MALDI targets and / or MPs will now be analysed using ESI-MS/MS, LC-ESI-MS/MS, MALDI MS or MALDI-MS/MS or a combination thereof. The instruments will yield raw data which is usually enormous and of different proprietary formats. Proteios should not import the raw data, but provide links to it. After the spectrum acquisition software is used that extract peak lists in different formats. There are a number of different peak list formats, Proteios should be able to import mzData and PKL at least. Preferably also ProteinLynx Global Server and DTA files. Different kinds of MS use cases here. Many files are generated and thus batch imports or daemon importers must exist. Since the peak lists will not contain any information about which MP or MALDI target they came from we are using naming conventions so that file name can be partly used for tracking of files. More information on file naming here MSFileNames.
Proteios should be able to export the peaklists in mzData or PKL format again. Thus Proteios can be used for the file format conversion.
Finally protein identification is performed using different software. For MALDI MS data PIUMS and Mascot search results importers have to exist. For MS/MS data there must be Mascot and X!Tandem importers. Sequest import would also be useful. Peak lists can be analysed several times with different settings or with different software.
The basic gel report report which proteins have been identified in each spot and show scores and other things needed for publication in MCP,JPR or Proteomics
