The Sequest HT node calculates XCorr scores for peptide matches and provides the peptide matches having the best XCorr score for each spectrum. It calculates the XCorr value for every peptide candidate.
You can specify one or more FASTA files for Sequest HT to search. When you specify the names of multiple FASTA files to search, Sequest HT searches these FASTA files as if they were combined into one FASTA file. If the application finds the same PSM in multiple FASTA files, it counts that PSM as only one PSM.
You can use the Protein Marker node in the consensus workflow to find in which FASTA file the application found a specific protein. For more information, see Protein Marker node and Working with grouping and quantification.
The following table describes the parameters for the Sequest HT node.
Parameters | Description |
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Protein Database | Specifies the names of the FASTA databases to search. To select one or more databases, select the check boxes next to the database names. The names of the selected databases are separated by colons in the Protein Database box. There is no limit to the number of FASTA databases that you can select. |
Enzyme Name | Specifies the reagent used for protein digestion. After you select your enzyme, you can also specify the type of enzymatic cleavage to consider (fully enzymatic, partially enzymatic, limited to C terminal, or limited to N terminal). To add to the list of enzymes, see Work with cleavage reagents. The available enzymes are the following:
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Max. Missed Cleavage Sites | Specifies the maximum number of missed cleavage sites per peptide to consider during the digest. Range: 0–12; default: 2 |
Min. Peptide Length | Specifies a peptide’s minimum sequence length. Range: 4–144; default: 6 |
Max. Peptide Length | Specifies a peptide’s maximum sequence length. Range: 4–150; default: 150 |
Max. Number of Peptides Reported | Specifies the maximum number of peptides reported per spectrum. Range: 1–100; default: 10 |
Precursor Mass Tolerance | Specifies the mass tolerance used for finding peptide candidates. You can specify the units (Da, mmu, or ppm) for the precursor mass tolerance value. Range: 0.0001–5.0 Da or 0.01–5000 ppm; default: 10 ppm |
Fragment Mass Tolerance | Specifies the mass tolerance used for matching fragment peaks. You can specify the units (Da or mmu) for the fragment mass tolerance value. Range: 0.02–2.0 Da; default: 0.6 Da |
Use Average Precursor Mass | Determines the type of mass to use for matching the precursor.
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Use Average Fragment Mass | Determines the type of mass to use for matching the peptide fragments.
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Use Neutral Loss a Ions | Determines whether Sequest HT uses neutral losses from a ions for spectrum matching.
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Use Neutral Loss b Ions | Determines whether Sequest HT uses neutral losses from b ions for spectrum matching.
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Use Neutral Loss y Ions | Determines whether Sequest HT uses neutral losses from y ions for spectrum matching.
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Use Flanking Ions | Determines whether Sequest HT adds flanking ions, which are two ions that it adds to each base ion to calculate a theoretical spectrum for each peptide candidate. These ions help to offset low-resolution spectra and broad peaks. The masses of the flanking ions are the base ion mass plus or minus 1 Da.
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Weight of a Ions | Uses a ions for spectrum matching with this relative factor. This value specifies the relative weight of a ion peaks when the theoretical spectra are generated. Range: 0.0–1.0; default: 0 |
Weight of b Ions | Uses b ions for spectrum matching with this relative factor. This value specifies the relative weight of b ion peaks when the theoretical spectra are generated. Range: 0.0–1.0; default: 1 |
Weight of c Ions | Uses c ions for spectrum matching with this relative factor. This value specifies the relative weight of c ion peaks when the theoretical spectra are generated. Range: 0.0–1.0; default: 0 |
Weight of x Ions | Uses x ions for spectrum matching with this relative factor. This value specifies the relative weight of x ion peaks when the theoretical spectra are generated. Range: 0.0–1.0; default: 0 |
Weight of y Ions | Uses y ions for spectrum matching with this relative factor. This value specifies the relative weight of y ion peaks when the theoretical spectra are generated. Range: 0.0–1.0; default: 1 |
Weight of z Ions | Uses z ions for spectrum matching with this relative factor. This value specifies the relative weight of z ion peaks when the theoretical spectra are generated. Range: 0.0–1.0; default: 0 |
Max. Equal Modifications Per Peptide | Specifies the maximum number of times that one dynamic modification can be found in a single peptide. Range: 0–8; default: 3 |
Max. Dynamic Modifications Per Peptide | Specifies the maximum number of differential modifications per peptide. Range: 0–12; default: 4 |
Dynamic Modification | Specifies the dynamic modifications used during the search. The available modifications are defined in the Chemical Modifications view on the Administration page. Default: None |
Dynamic Modification (Peptide Terminus) | Specifies the dynamic modifications at the ends of a peptide. |
N-Terminal Modification | Specifies the dynamic modification at the N terminus of a peptide. From the list, select a modification. The application automatically selects and populates the modification site. The list is composed of amino acids that you select on the Chemical Modifications page. Default: None |
C-Terminal Modification | Specifies the dynamic modification at the C terminus of a peptide. From the list, select a modification. The application automatically selects and populates the modification site. The list is composed of amino acids that you select on the Chemical Modifications page. Default: None |
Dynamic Modifications (Protein Terminus) | Specify the dynamic modifications at the ends of a whole protein. This protein is composed of several peptides that are cleaved by the cleavage enzyme before the analysis is done. |
N-Terminal Modification | Specifies the dynamic modification at the N-terminus of a protein. From the list, select a modification. The application automatically selects and populates the modification site. The list is composed of amino acids that you select on the Chemical Modifications page. Default: None |
C-Terminal Modification | Specifies the dynamic modification at the C-terminus of a protein. From the list, select a modification. The application automatically selects and populates the modification site. The list is composed of amino acids that you select on the Chemical Modifications page. Default: None |
Static Modifications | Specify the static modifications used during the search. The available modifications are defined in the Chemical Modifications view on the Administration page. Default: None |
Peptide N-Terminus | Specifies the static modification for the N-terminal of the peptide used during the search. Default: None |
Peptide C-Terminus | Specifies the static modification for the C-terminal of the peptide used during the search. Default: None |
Static Modification | Applies the same specific mass to all occurrences of that named amino acid, as in an exhaustive chemical modification. You define the available modifications in the Chemical Modifications view on the Administration page, which you open by choosing Administration > Maintain Chemical Modifications. Default: None |