The Identification subpage of the processing method editing wizard contains six sections: Peptide Identification, Advanced Search, Disulfide Search, Reduced LC/MS Run, Select Protease, and Delete or Add New Protease.
| No. | Description | No. | Description |
|---|---|---|---|
1 | Parameters tab (processing method editing wizard) | 5 | Disulfide Search parameters |
2 | Subpage navigation bar | 6 | Reduced LC/MS Run parameters |
3 | Peptide Identification parameters | 7 | Select Protease parameters |
4 | Advanced Search parameters | 8 | Delete of Add New Protease area |
The table below describes the parameters on the Identification subpage.
Parameter | Description |
|---|---|
Peptide Identification | |
Search by Full MS Only | Indicates the type of data that application will use to search.
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Use MS/MS | Specifies the type of data to process.
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Maximum Peptide Mass | Specifies the maximum peptide mass to be identified. Increase this value to search for disulfide bonds in non-reduced samples.
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Mass Accuracy (ppm) | Specifies the maximum deviation (ppm) between the mass of a theoretical peptide and the calculated mass of a particular ion used to identify a peptide.
For non-targeted analyses, the application does not strictly enforce this parameter so that it identifies more peptides. For targeted analyses, the application strictly enforces this parameter so that it does not identify peptides outside of the mass tolerance. |
Minimum Confidence | Specifies the minimum confidence level for a reported peptide assignment on a 0-to-1 scale, with 1 being the highest confidence.
If “Yes” is selected in the Search by Full MS Only field, the minimum confidence will be set to 0.00. If you create custom glycans and assign them as side chain modifications to a protein sequence used in the analysis, you must set this parameter to 0.00; otherwise, the application will not identify peptide components with these custom glycan modifications. |
Maximum Number of Modifications for a Peptide | Read-only. Specifies the maximum number of modifications allowed for each peptide; previously specified in the Assign Variable Modifications area of the Sequence Editor page. See Manage variable modifications.
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Advanced Search | |
Enable Mass Search for Unspecified Modifications (checkbox) | Select the Enable Mass Search for Unspecified Modifications checkbox to allow the application to perform a mass search for unspecified modifications to the peptide. |
Mass Changes for Unspecified Modifications | Defines the range of changes to the mass of a peptide resulting from an unspecified modification. Only integer values are possible.
Read-only if the Enable Mass Search for Unspecified Modifications checkbox is not selected. For full characterization of a target protein, you must identify unspecified modifications. To identify an unspecified modification, the application applies a mass change within the defined range to the mass of an unknown peptide and attempts to match the modified mass to the mass of an identified peptide. If the application finds a match, but cannot determine the exact modification site, it places a tilde (~) mark before the modification site to indicate the approximate location of the unspecified modification. For example, an unspecific modification on a peptide is denoted at ~C310-57.0212. This indicates the loss of 57.0212 Da near Cys-310, which suggests an incomplete alkylation. |
Glycosylation | Read-only. Specifies the type of glycosylation to apply to the N-/O-linked glycans previously specified in the N, O Glycan section of the Glycosylation area of the Sequence Editor page. See Manage glycosylations. Default: None |
Search for Amino Acid Substitutions | Determines how the application searched for amino acid substitutions.
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Enable Residue Deletion (checkbox) | Select the Enable Residue Deletion checkbox to allow the application to search for possible peptide sequences that may be missing a single amino acid residue at any position. |
Disulfide Search | |
Perform Disulfide Bond Search | Determines whether the application performs a search for disulfide bonds.
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Allow Free Cys (checkbox) | Determines whether the application allows free cysteine (C) residues to exist in the peptide sequence. Read-only if Perform Disulfide Bond Search is not selected. |
Maximum Number of Hits | Specifies the maximum number of disulfide-linked peptides the application finds before stopping the search for disulfide bonds in the protein sequence.
Read-only if Perform Disulfide Bond Search is not selected. |
Maximum Number of Disulfide Bonds | Specifies the maximum number of disulfide bonds identified by the application during the search for disulfide bonds in the protein sequence.
Read-only if Perform Disulfide Bond Search is not selected. |
Maximum Number of Identical Chains in the Molecule | Specifies the maximum number of identical chains in the protein. For example, if the protein is a disulfide-linked homo-dimer, this parameter should be set to 2.
Read-only if Perform Disulfide Bond Search is not selected. |
Reduced LC/MS Run | |
Reduced LC/MS Run (dropdown menu) | Specifies the name of the reduced raw data file in the analysis (if available). If you select “Yes” for Perform Disulfide Bond Search, specifying the reduced raw data file helps the application identify disulfide-linked peptides. If you have both reduced and non-reduced raw data files for an analysis, you can process both files to generate more reliable disulfide-bond assignments. To do this, upload both the reduced and non-reduced raw data files on the Peptide Mapping Analysis page and when editing the processing method, select the name of the reduced raw data file in the Reduced LC/MS Run box on the Identification subpage of the Parameters page. |
Select Protease | |
Select Protease (dropdown menu) | Specifies the protease assigned to the currently open processing method. The application provides a list of default proteases in the Select Protease dropdown menu. If the appropriate protease is not listed, you can add a custom protease before assigning it to the processing method.
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N-Term and C-Term | Read-only. Displays the activity of the selected protease at the N- and C-terminals. If the “Nonspecific” protease option is selected, these boxes are empty. |
Specificity | Defines the level of protease specificity for the protease assigned to the currently open processing method.
Specificity levels High, Medium, and Low correspond to confidence factors that the application uses to determine the final confidence score of component identification. The difference between these levels can impact your results when two or more components match the spectrum, but only one of the components adheres to the specificity requirements of the selected protease. Thus, if your sample includes many miscleavages, you might choose to use Medium or Low. If the specificity is set to Strict, the application only identifies components that exactly match a theoretical peptide under the specific protease conditions assigned. |
Delete or Add New Protease | |
Select Protease (dropdown menu) | Specifies the protease you want to edit (if adding a new protease) or delete. |
Protease Name | Assigns a name to the protease to be edited (if adding a new protease) or deleted. If adding a new protease, type the name of the new protease in this box. |
N-Term and C-Term | Specifies the activity of the selected protease at the N- and C-terminals. If adding a new protease, enter the appropriate N-/C-terminals activity for your protease into these boxes. |
In the Delete or Add New Protease section of the Identification page, you can add, delete, or edit user-created custom proteases, as described in the table below.
Task | Actions |
|---|---|
Add a custom protease |
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Delete a custom protease |
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Edit an existing custom protease |
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NOTE
The protease options "Nonspecific", “No enzyme”, or any protease with cleavage sites at both the N- and C-terminals are not supported for theoretical digestion at this time.
Thus, if you want to enable the Customize Results subpage of the processing method editing wizard, do not use these protease options on the Identification subpage.