An open source tandem mass spectrometry (MS/MS) sequence database search tool. The comet is a search engine developed by Jimmy Eng, who is an author of SEQUEST. In the IP2, we have added additional features such as heavy/medium search, graphical user interface, cluster compatibility, and more.

From experiment page, click the Comet search link to open search parameter page.

Figure 7.2.1: Link and button control to start Comet search

The user needs to define search parameters before running Comet. IP2 will automatically save the search parameters for subsequent searches within same project. In the search parameters, the user also needs to define the computational resource as either “cluster” or “cloud” as shown below:

1. Computational resource
Select either cluster or cloud for “Computational resource”.

Figure 7.2.2: Comet search parameter “computational resource”

2. Basic parameters:

Figure 7.2.3: Basic Comet Parameters

Search Name
User defined search name. When you run multiple searches on same data, you can give different name to each search.
User
Select a user to list databases owned by his/her account
Protein database
Choose an appropriate protein FASTA database. If protein database is not available, click database and add the new protein database.
Unit
units applied to the peptide_mass_tolerance parameter.
Precursor/Peptide mass toleranceUnit
Precursor mass tolerance
Fragment bin tolerance
bin size associated with fragment ions
Ions to search
Fragment ions to search on
mass range
mass range of peptides to search

3. Enzyme parameters:

Figure 7.2.4: Enzyme parameters

Specificity (terminal miscleavage)
the number of enzyme termini a peptide must have
Max num internal miscleavage
Maximum number of internal miscleavage
Enzyme Selection
The first column of the parameter definition is the enzyme number. This number list must start from 0 and sequentially increase by 1. The second column is the enzyme name; no spaces are allowed in this name field. The third column is the digestion sense i.e. a value of 0 specifies cleavage N-teriminal to (before) the specified residues in column 4 and a value of 1 specifies cleavage C-terminal to (after) the specified residues in column 4. Column 4 contains the residue(s) that the enzyme cleaves at. Column 5 contains the flanking residue(s) that negate cleavage.

4. Differential modification parameters:

Figure 7.2.5: Differential modifications

Maximum number of internal diff mods
maximum number of residues that can be modified in a peptide C terminus mass shift
a variable modification to peptide’s c-terminus
N terminus mass shift
a variable modification to peptide’s n-terminus
mass shift and residues
Residue specific differential modification. Specify mass shift and residues e.g., 79.9663 STY for phosphorylation on S or T or Y residues 42.0106 K for Acetylation on K 114.042927 K for ubiquitinylation on K 15.9949 M for Oxidation on M -18.0289 STY neutral loss for ms3 diff search

5. Static/fixed modifications:

Figure 7.2.6: Comet static/fixed modifications

N-term peptide static modification
a static modification to the n-terminus of all peptides
C-term peptide static modification
a static modification to the c-terminus of all peptides
N-term protein static modification
a static modification to the n-terminal peptide of each protein entry
C-term protein static modification
a static modification to the c-terminal peptide of each protein entry
C-term protein static modification
a static modification to the c-terminal peptide of each protein entry
Amino acid residue specific static modification
Residue specific static modification. Specify mass shift and residues

6. Processing spectra:

Figure 7.2.7: Processing spectra

Max. # of scans in split spectral files
Maximum number of spectra in split spectral files before submitting search jobs

7. Labeling Search:

Figure 7.2.8: Labeling Search

Would you like to include heavy isotopic labeling search
Please check yes if you want to perform heavy search together with regular search. It is recommended to use this option to maximize identification
Metabolic labeling type
For metabolic labeling with selected amino acids, e.g., SILAC, you will need to specify mass shifts of labeled amino acid residues. For N15 search, there is no need to specify mass shift of each amino acid residue. IP2 will automatically do it for you
heavy mod medium mod
click ‘add static mod for heavy search’ to define residue and mass shift

8. Basic DTASelect 2.0 Parameters:
Minimum number of peptide per protein (-p)
Set minimum peptides per protein. E.g., 2 to accept proteins with at least 2 peptides passed filtering.
Minimum number of tryptic end per peptide (-y)
Accept peptides that fulfill the specified enzyme specificity. 0 to include all peptides regardless enzyme specificity; 1 to include peptide with at least 1 tryptic end; 2 to include only peptides with both ends are tryptic
False positive rate
Choose Spectrum(—fp) to specify spectral (PSM) level false positive rate; choose Protein(—pfp) to specify protein level false positive rate; choose Peptide(—sfp) to specify peptide level false positive rate. The value should be between 0 and 1. For example, 0.05 means 5% false positive rate.
Precursor delta mass cutoff (-DM)
Precursor delta mass cutoff in ppm. For example, 5 means accept peptides that have delta masses no more than 5 ppm

Figure 7.2.9: Basic DTASelect Parameters

Advanced DTASelect 2.0 Parameters:
Peptide modification requirement (-m)
Display peptide based on the modification status. 0 to include modified peptide only; 1 to include peptide regardless the modification status; 2 to include only unmodified peptides
Best peptide FP threshold (-tfp)
For each protein, require to have at least one peptide at the specified false positive or confidence level, e.g., 0.01 or 0.001. 0.001 mean a protein has to have one peptide with confidence level 0.999.
Best peptide delta mass threshold (-tDM)
For each protein, require to have at least one peptide with ppm delta mass not greater than this value, e.g., 5 will require each protein to have at least one peptide with delta value <= 5 ppm.
Spectra display mode (-t)
With the –t option, you can specify how the peptide spectrum matches should be displayed in the DTASelect results. Setting it to 0 to include all spectra for each sequence; 1 to include one spectrum per salt step for each peptide sequence; 2 to include only one spectrum for each peptide sequence
statistics with delta mass (—mass)
Using delta mass in statistics. With this option, DTASelect will give higher scores to peptide hits with better delta mass values.
statistics with modification (—modstat)
With –modstat option, DTASelect2 will score modified and unmodified peptides separately to ensure specified false positive rate in each categories.
statistics with tryptic status (—trypstat)
With –trypstat option, DTASelect will score none, half and full tryptic peptides separately to ensure user specified false positive rate in each categories.
Include subset proteins (-in)
With –in option, DTASelect2 will include subset proteins in the result files. By default, DTASelect2 results do not include subset proteins. If protein A is identified with 3 peptide a, b and c, and protein B is identified with 2 peptides a and b, then protein B is a subset protein of protein A.
Protein ID filter (-e)
With –e option, DTASelect will remove proteins with IDs starting with this string
Peptide sequence filter (-Sic)
With –Sic option, DTASelect2 will only accept peptide sequences must contain all of these characters
Additional DTASelect options
Put all the additional options that are not listed above in this box. e.g., —MC 1 to specify max number of missed cleavage sites to 1. Different options should be separate with a space character. All the DTASelect2 options can be found at http://fields.scripps.edu/pub/dtaselect2options.txt

Figure 7.2.10: Advanced DTASelect Parameters

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