The IMP-ptmRS node calculates the modification site probabilities for all modifications used in the search that are not on terminals. The node adds the following columns to the PSMs page of the result file:
- ptmRS Best Site Probabilities column
- ptmRS Modification Site Probabilities column
- ptmRS Binomial Peptide Score column
- ptmRS Isoform Confidence Probability column
- ptmRS Isoform Group Report column
Tn the processing workflow, the ptmRS node provides a confidence measure for the localization of phosphorylation and any other modifications in peptide sequences. You can use it with all common fragmentation techniques, such as CID, ETD, and HCD, and peptide identifications from all available database search engines.
This node uses parallelized calculations with multiple processor cores, when available, to improve performance. It also determines the optimal number of peaks to consider for localization of PTM sites for each m/z window individually, which increases the sensitivity of site localization for CID data.
Depending on the applied fragmentation technique, the ptmRS node uses different fragment ion types for scoring to provide the highest sensitivity.
- For CID data, it scores only singly and doubly charged b and y ions.
- For analysis of HCD spectra, it also considers neutral loss ions.
- For localizing PTM sites in ETD spectra, it exclusively considers singly charged c, z, and y+H ions.
The input to ptmRS is a list of filtered MS/MS fragment spectra with corresponding peptide identifications.
The following table describes the parameters for the ptmRS node.
Parameter | Definition |
|---|---|
PhosphoRS Mode | Determines whether to assign modification site scores to the target amino acids of only phosphorylation modifications or of all modifications. A high score indicates that the modification is placed, or localized, on the corresponding target.
|
Report Only PTMs | Determines whether the ptmRS node performs site localization scoring for all modifications or only for those modifications marked as post-translational in the Chemical Modifications view on the Administration page. The site localization score is a measure of confidence that a modification resides at a specific site on a peptide.
|
Use Diagnostic Ions | Determines whether to use diagnostic ions to rule out potential isoforms and therefore limit the search space.
|
Use Fragment Mass Tolerance of Search Node | Determines whether to use the fragment mass tolerance set in the search node or the fragment mass tolerance set by the Fragment Mass Tolerance parameter for matching fragment ion peaks.
|
Fragment Mass Tolerance | Sets the mass tolerance used for matching fragment ion peaks. Range: 0.0001 Da–1.0 Da; default: 0.5 Da You can also set the tolerance in mmu, for example, 0.1–1000 mmu. |
Note The set mass tolerance is only used when you set the Use Fragment Mass Tolerance of Search Node parameter to False. | |
Consider Neutral Loss Peaks for CID, HCD, and EThcD | Determines whether to consider neutral loss peaks for scoring when analyzing CID, HCD, or EThcD (b/y) data. By default, it scores such peaks for HCD and EThcD (b/y) spectra only because their inclusion in the analysis of CID data leads to an decrease in specificity of phosphorylation site localization. The application never scores neutral loss peaks for ETD and ECD spectra.
|
|
|
Maximum Peak Depth | Specifies the maximum peak depth. The ptmRS algorithm becomes more sensitive if it considers more peaks in each 100 Da window of the spectrum, but the increase in sensitivity comes with a decrease in speed and an increase of false positive matches. Range: 3–no maximum; default: 8 |
Use a Mass Accuracy Correction | Determines whether to correct a fragment ion mass distribution that is not uniform. False indicates that no correction is used.
|
Maximum Number of Position Isoforms | Specifies the highest number of position isoforms per peptide. The ptmRS node does not analyze peptides having more such position isoforms. This parameter greatly affects the ptmRS run time. Range: 0–no maximum (0 indicates no limit); default: 500 |
Maximum PTMs Per Peptide | Specifies the highest number of PTMs per peptide that can undergo any neutral molecular loss upon fragmentation. The ptmRS node does not analyze peptides having more such modifications. This parameter greatly affects the ptmRS run time. Range: 0–no maximum (0 indicates no limit); default: 10 |