For each spectrum file, the Spectrum Files RC node calculates a constant mass shift, in ppm, by performing a quick Sequest HT search and using the Fixed Value PSM Validator node to identify highly confident PSMs. The node calculates the PSM delta masses between theoretical and experimental mass, in ppm, and then calculates the mass shift curve as the median of the PSM delta masses. It can calculate a mass shift only when it can find at least 50 PSMs of high confidence.
The node can process MS/MS spectra with mixed activation types. It performs a separate search for each different activation type.
When you use the Spectrum Files RC in a workflow, the Spectrum Selector node uses recalibration factors to recalibrate MS1 precursor masses of the MS/MS spectra. All the nodes that follow the Spectrum Selector node in the workflow, such as Sequest HT, receive recalibrated spectra. The Feature Detection nodes and the Reporter Ions Quantifier node also use recalibrated LC/MS peaks, LC/MS features, and quantification spectra.
The Spectrum Files RC node is available only if you have a base license.
The following table describes the parameters for the Spectrum Files RC node.
Parameters | Description |
|---|---|
Protein Database | Specifies the names of the FASTA databases to search. |
Enzyme Name | Specifies the reagent used for protein digestion. You can only use full enzymatic digestion. Default: Trypsin (Full) |
Precursor Mass Tolerance | Specifies the mass tolerance used to find peak candidates. Range: 0.0001 Da–no maximum or Default: 20 ppm |
Fragment Mass Tolerance | Specifies the mass tolerance used for matching fragment peaks. Range: 0.02 Da–2.0 Da; default: 0.5 Da |
Dynamic Modification | Specifies the dynamic modification. The available modifications are defined in the Chemical Modifications view on the Administration page. You can specify phosphorylation as a dynamic modification for phospho-enriched samples so that the application finds enough initial identifications. |
Static Peptide N-Terminus | Specifies the static modification for the N-terminal of the peptide used during the search. The node universally applies static modifications to every instance of the specified residue or terminus. Default: None |
Static Peptide C-Terminus | Specifies the static modification for the C-terminal of the peptide used during search. The node universally applies static modifications to every instance of the specified residue or terminus. Default: None |
Static Modification | Specifies the same specific mass for all occurrences of a named amino acid. You can select any known or suspected modification. The node universally applies static modifications to every instance of the specified residue or terminus. Default: None |
Regression Model | Specifies whether to use a linear or non-linear regression over the retention time.
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Parameter Tuning | Determines how the application tunes the parameters for non-linear regression.
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