ProLuCID is a sensitive tandem mass spectra-based protein identification program developed in Yates laboratory at The Scripps Research Institute.
From the experiment page, click the ProLuCID search button to open search parameter page.
Figure 7.1.1: link to start ProLuCID search.
The user needs to define search parameters before running ProLuCID. IP2 will automatically save the search parameters for subsequent searches within the same project. In the search parameters, the user also needs to define the computational resource as either “cluster” or “cloud” as shown below (depending on your IP2 configuration)
Select either cluster or cloud for “Computational resource”.
Figure 7.1.2: ProLuCID search parameter: computational resource.
These are the basic ProLuCID search parameters:
Search name: User-defined search name.
User: Choose a user to display his/her databases. Users can use databases owned by other users.
Protein database: Choose appropriate protein FASTA database. If the protein database is not available, click the database and add the new protein database to IP2.
Fragmentation/activation method: Choose either CID or ETD (Choose CID for HCD data)
Precursor/peptide mass tolerance: If MS scan was done in the high-resolution mass analyzer, such as Orbitrap, select High resolution and then specify ppm precursor tolerance in ppm; if the instrument was set to select only the monoisotopic peak of the precursor, set the number of isotopic peaks to 1.
Otherwise, set the number of isotopic peaks to an integer greater than 1. For example, setting a number of isotopic peaks to 3 will consider the precursor peak as monoisotopic peak, M+1 peak (with one 13C in the peptide) and M+2 peak (with two 13C in the peptide)
If the MS scan was done in the low-resolution mass analyzer, such as LTQ, then click “low resolution” and specify “milli-AMU precursor tolerance” in milli-AMU. For example, 3000 for ±3 AMU
Fragment mass tolerance in ppm: if MS/MS spectra were acquired in low-resolution mass analyzer, such as LTQ, set Fragment mass tolerance in ppm to 400 to 600 ppm; On the other hand, if MS/MS spectra were acquired in high-resolution mass analyzer, such as Orbitrap, set Fragment mass tolerance in ppm to 5 to 100 ppm
Figure 7.1.3: Basic ProLuCID Parameters.
Choose “none” for no enzyme constraints; choose “one end” to consider candidate peptides with at least one end fulfill the enzyme specificity; choose “both end” to consider only candidate peptides with both ends fulfill the enzyme specificity
Max num internal miscleavage
Select either 0, 1, 2, 3 or unlimited number of peptide internal missed cleavage sites allowed in the candidate peptides
Specify the name of protease used to digest the sample
Specify the residues that the protease cut at, e.g., KR for trypsin
Choose cterm if the enzyme cleaves at the C terminus of specified residues; choose nterm if the protease cut at the N terminus of the specified residues
Figure 7.1.4: ProLuCID enzyme specificity
N-term static modification
Specify the mass shift of peptide N terminus static/fixed modification. For example, 225.1558 TMT 2-plex on peptide n-term; 229.1629 for TMT 6-plex on peptide n-term; 144.1021 for iTRAQ peptide n-term
C-term static modification
Specify the mass shift of peptide C terminus static/fixed modification.
Amino acid residue specific static modification
Specify residue-specific static modification, mass shift and residues e.g., 57.02146 C for Carbamidomethylation; 225.1558 K for TMT 2-plex on K; 229.1629 K for TMT 6-plex on K; 144.1021 K for iTRAQ on K
Figure 7.1.5: ProLuCID fixed/static modifications
- a Maximum number of internal diff mods* Specify the maximum number of peptide internal differential modifications to be considered in the search. In order to consider any internal variable modification, it has to be set to an integer greater than 0.
Differential/variable modification Specify Residue specific differential modification, 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
Figure 7.1.6: Differential/variable modifications
Metabolic 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.
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 a mass shift of each amino acid residue. IP2 will automatically do it for you.
What is the metabolic labeling efficiency? Specify the labeling efficiency. This is important for N15 labeling.
Specify mass shifts of selected labeled amino acids You need to specify the mass shift of labeled (heavy) amino acid residues. For example, 10.0083 R for SILAC heavy R residue 6.0201 K or 8.0142 K for K residue. Please note, for N15 metabolic labeling, you do not need to specify the mass shift of each amino acid residue. IP2 will automatically do it for you. If you have 3-plex labelings like SILAC or Dimethyl, you can specify medium labeling values.
Figure 7.1.7: Metabolic Labeling Parameters
IP2 also provide advanced ProLuCID parameters for the search. The form can be displayed when user clicks the Tick.png below the “Metabolic Labeling Search” section.
Advanced ProLuCID parameters:
Primary score type
Specify the primary score type. XCorr should work well for most of the cases. Probability score can be chosen if the MS/MS were done at high resolution.
Secondary score type
Choose the secondary score type. Secondary score type should be different from Primary score type.
XCorr: cross-correlation score between theoretical and experimental peaks
Probability: a probability score based on preliminary score
z-score: a z-score for all peptide candidates based on XCorr score
p-value: a p-value for all peptide candidates based on XCorr score
e-value: an e-value for all peptide candidates based on XCorr score
Are MS/MS spectra deisotoped and decharged
Check yes if the MS/MS spectra are deisotoped and decharged
Multistage Activation Mode
0 to consider only non-neutral loss peaks 1 to consider both neutral loss and non-neutral loss peaks 2 to consider only neutral loss peaks
Minimum peptide length
Set the minimum length of candidate peptides. A peptide with a length smaller than the minimum peptide length will not be considered.
Candidate peptide threshold Set number of candidate peptides for final scoring. The default value is 500. However, a larger number (e.g., 5000 or 50000) may be needed if the redundancy of the protein database is extremely high.
Peptide terminal differential/variable modifications
Peptide N-term diff mods: Peptide N-term diff mods, e.g., 42.0106 for Acetylation, 57.02146 for n-term Alkylation, -17.0265 for Loss of ammonia, 43.0058 for Carbamylation
Peptide C-term diff mods: Peptide C-term diff mods, e.g., -18.01057 for loss of water
Figure 7.1.8: Advanced ProLuCID Search Parameters
Basic DTASelect 2.0 Parameters:
h4. Figure 7.1.9: Basic DTASelect Parameters
Minimum number of peptide per protein (-p)
Set minimum of 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
Advanced DTASelect 2.0 Parameters:
h4. Figure 7.1.10: Advanced DTASelect Parameters
Peptide modification requirement (-m)
Display peptide based on the modification status. 0 to include modified peptide only; 1 to include peptide regardless of 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 a specified false positive rate in each category.
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 category.
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 peptides 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 the max number of missed cleavage sites to 1. Different options should be separated with a space character. All the DTASelect2 options can be found at http://fields.scripps.edu/pub/dtaselect2options.txt
For dimethyl labeling, refer to Figure 7.1.11