Adding PTM Analysis to a Workflow - Proteome Discoverer Software - Adding PTM Analysis to a Workflow - Proteome Discoverer Software - Proteome Discoverer Software

To focus on studying the biologically relevant post-translational modifications of proteins, you can add PTM analysis to workflows.

Modifications of Proteins on the PSMs Page

The Proteome Discoverer application displays the positions of the identified modifications in a protein the same way that it displays the positions of a peptide in a protein. The way it displays peptide modifications is described in the Modifications of peptides on the Peptide Groups page topic in the Proteome Discoverer Help.

The application creates two different modification columns. One modification column contains only the best possibilities for the modifications. The other contains all possible positions. The Modification Sites Shown parameter of the PSM Grouper node determines whether the application shows one or both of these columns. The column not shown is still available, and you can display it by using the Field Chooser. (See the Display the columns on the result pages in the Proteome Discoverer Help.)

For detailed information on the parameters of the PSM Grouper node, see the PSM Grouper node in the Proteome Discoverer Help and the decision tree for the modification display and the accompanying examples.

You can also use the PSM Grouper node’s Site Probability Threshold parameter to determine whether the application should display only sites with a minimal site probability score.

Modifications of Proteins on the Proteins Page

To restrict the concatenated list of modifications in the Modification column on the Proteins page, set these parameters:

• The Site Probability Threshold parameter of the PSM Grouper node
• The Report Only PTMs parameter of the Peptide in Protein Annotation node to show only post-translational modification sites

You can also use the Peptide in Protein Annotation node’s Minimum PSM Confidence parameter to restrict the modifications shown to those with a minimal confidence level for the PSMs that identified the modifications.

Depending on the protein, the list of modifications can become long. When you are interested in only one type of modification, use the Modification parameter (under Modifications in Protein) to display a set of seven (maximum) selected modifications in individual columns. For each of these specified modifications, the application creates two new columns. One column contains the modifications and all sites where the modification is found, and the second column shows how many times this modification occurs in the protein.

Special Processing Workflow Nodes and Node Settings

You can use one of the processing workflow templates that includes IMP-ptmRS, for example, PWF_Hybrid_SequestHT_MSAmanda_Percolator_ptmRS.pdProcessingWF, or add the following nodes in the processing workflow:

• Spectrum Selector
• PSM validation node, such as Fixed Value PSM Validator, Percolator, or Target Decoy PSM Validator
• IMP-ptmRS node attached to a PSM validation node, for example, the Target Decoy PSM Validator node

Special Consensus Workflow Nodes and Node Settings

To increase the information in results, add the following nodes to consensus workflow:

• The Peptide Isoform Grouper node, which adds an additional results page specifying the number of isoforms that are found for each peptide group. (The node connects to the Peptide and Protein Filter node.)
• The Modification Sites node, which adds an additional results page that contains information on all the PTM sites in the result. (The node connects to the Protein Grouping node.)
• The Peptide in Protein Annotation node, which provides additional information about the modifications on the protein level. The Protein Positions for Peptides parameter of the node determines whether the Proteome Discoverer application displays the peptide or PSM position for all proteins or only for the master proteins.

The consensus workflow CWF_Basic_ModificationAnalysis includes the above nodes and is a good workflow to use for PTM analysis. However, if you need quantification, add the appropriate quantification nodes to this default workflow.

Further Information

The IMP-ptmRS node generates the PSM-specific modification site data. That is, the IMP-ptmRS node calculates modification site probabilities and makes them available in the Protein Identification Details view. For more information on this view, see Use the Protein Identification Details view.

For more information on the IMP-ptmRS node, see the IMP-ptmRS node in the Proteome Discoverer Help.

For more information on the Peptide in Protein Annotation node, see the Peptide in Protein Annotation node in the Proteome Discoverer Help.