The INFERYS Rescoring node receives the results from Sequest HT, automatically determines the appropriate HCD or CID collision energy, produces the theoretical MS/MS spectra for each target and decoy PSM including lower ranked matches, and calculates new figures-of-merit for input into Percolator.
The INFERYS Rescoring only supports HCD and CID data of peptides with carbamidomethyl cysteine, N-terminal TMT and TMTpro, TMT and TMTpro lysine, and optional oxidized methionine. INFERYS Rescoring can predict peptide sequences from 7-30 amino acids with precursor charge states from 1-6+ and do not need to be trypsin specific.
To include INFERYS Rescoring in a workflow, it is best to start with one of the default processing workflow templates due the requirement for specific settings in the Spectrum Selector and Sequest HT nodes. These settings are different than the default settings for each of these nodes and are also different than any of the other default workflows.
Special Processing Workflow Nodes and Node Settings
There are several default processing workflows that include the INFERYS Rescoring node:
PWF_Hybrid_MPS_SequestHT_INFERYS_Rescoring_Percolator.pdProcessingWF
PWF_Hybrid_MPS_SequestHT_INFERYS_Rescoring_Percolator_2stage.pdProcessingWF
PWF_Hybrid_TMT_Sequest HT_INFERYS_Rescoring_Percolator.pdProcessingWF
PWF_Hybrid_TMTpro_Sequest HT_INFERYS_Rescoring_Percolator.pdProcessingWF
PWF_Tribrid_TMT_Quan_SPS_MS3_SequestHT_INFERYS_Rescoring_Percolator.pdProcessingWF
PWF_Tribrid_TMT_SPS_MS3_RTS_Sequest HT_INFERYS Rescoring_Comet_Percolator.pdProcessingWF
PWF_Tribrid_TMTpro_SPS_MS3_RTS_Sequest HT_INFERYS Rescoring_Comet_Percolator.pdProcessingWF
PWF_Tribrid_TMTpro_SPS_MS3_Sequest HT_INFERYS Rescoring_Comet_Percolator.pdProcessingWF
PWF_Tribrid_TMTpro_Quan_SPS_MS3_SequestHT_INFERYS_Rescoring_Percolator.pdProcessingWF
We have included INFERYS Rescoring workflows to support basic peptide identification and TMT and TMTpro workflows.
The 2-stage processing workflow includes a second Sequest HT search to identify peptides with post-translational modifications that are not supported by INFERYS Rescoring.
You can modify these INFERYS Rescoring workflows further to support label-free and precursor quantification by adding a Minora Feature Detector node or reporter ion quantification by adding a Reporter Ions Quantifier node.
Special Consensus Workflow Nodes and Node Settings
Choose the consensus workflow depending on the type of quantification methods that were added to the Processing workflow. If no quantification is performed, any of the CWF_Basic or the CWF_Comprehensive_Enhanced_Annotation workflows are suitable. If quantification nodes were added to the workflows, choose the appropriate consensus workflow for that quantification method. A description of these workflows is in the sections immediately preceding this one.