Scaffold reads files from Proteome Discoverer and numerous quantitative options are supported. However, PD must be configured properly to ensure Scaffold has access to both identification and quantitative data. To begin a few notes:
- Scaffold reads the MSF files created by Proteome Discoverer. For versions 2.x both the MSF and the PDStudy file must be in the same directory.
- Make sure to Save All in PD before loading files into Scaffold
- When running the Daemon, Scaffold needs access to the PARAMS file instead of the PDStudy file. Make sure this is in the same directory as the MSF files.
Note, at this time Scaffold does not support custom quantitative methods created in PD. Please use the default methods for iTRAQ, TMT or SILAC quantification.
Scaffold also does not support loading one study that was searched using multiple instances of the same search engine node. For example, searching with two Sequest nodes is not supported while searching with one Sequest and one Mascot node is supported.
Scaffold 4.8.1 introduced prefiltered mode which allows users to bypass Scaffold’s built in probability model and filter your data using the search engine instead. Use the Percolator node in the processing workflow and set the FDR cutoff to what you consider an acceptable value.
Starting in Scaffold 4.11.0 you will be able to threshold your data using the dropdowns in the Samples view using Percolator-derived probabilities. Otherwise, when you use prefiltered mode you will not be able to adjust the probability thresholds. If using an earlier version of Scaffold it is recommended to then set a custom peptide threshold using the Edit menu.
Scaffold supports multiple types of quantitative experiments when loading files from Proteome
Discoverer including iTRAQ and TMT, SILAC, and precursor intensity quantitation. Each type of
experiment requires a different quantitative node in the processing workflow:
Precursor Intensity Quantitation
The following is an example processing workflow for precursor intensity quantitation
The following is an example of the processing workflow for SILAC quantitation
The Precursor Ions Quantifier node is added to a SILAC workflow. Make sure you choose the proper SILAC Quantitation Method and modifications for your experiment
Reporter Ion Quantitation
The following is an example processing workflow for reporter ion quantitation.
The Reporter Ions Quantifier node is added to an iTRAQ or TMT workflow. Make sure you choose the proper Quantitation Method and modifications for your experiment.