What it does

PeptideShaker is a search engine for interpretation of proteomics identification results from multiple search engines, currently supporting X!Tandem, MS-GF+, MS Amanda, OMSSA, MyriMatch, Comet, Tide, Mascot, Andromeda and mzIdentML.

http://compomics.github.io/projects/peptide-shaker.html

Outputs

zip

When choosing zip option, all other outputs are compressed and included into the zip file and, by default, no other file is shown in the Galaxy history. This last behaviour may be changed in order to have all other outputs also available in the history.

psdb

psdb is the native format used by PeptideShaker. It contains all the information required by PeptideShaker to run.

mzIdentML

PeptideShaker can load results from virtually any identification algorithm in the mzIdentML format as long as the minimal peptide to spectrum match information is present in the file.

The following is required:

Spectrum file format has to be mgf.
Each PSM has a score or e-value as a PSM score CV term.

Follow-up analysis

Spectra

Exports the spectra according to the category of the PSMs:

Spectra of Non-Validated PSMs
Spectra of Non-Validated Peptides
Spectra of Non-Validated Proteins
Spectra of Validated PSMs
Spectra of Validated PSMs of Validated Peptides
Spectra of validated PSMs of Validated Peptides of Validated Proteins.

Exported results are in mgf format.

Proteins: Accession numbers

Export the protein accessions according to the category of the proteins:

Main Accession of Validated Protein Groups
All Accessions of Validated Protein Groups
Non-Validated Accessions

Exported protein accessions are text format as TXT.

Proteins: Sequences

Exports the protein details according to a category of proteins:

Main Accession of Validated Protein Groups
All Accessions of Validated Protein Groups
Non-Validated Accessions.

Exported protein details are in fasta format.

Proteins: Proteoforms

Exports all possible proteoforms of the validated proteins.

Exported results are in text format as TXT. Results file can be easily imported and used by PathwayMatcher tool.

Label free quantification

Exports the spectra from different categories of PSMs according to the export type:

Validated PSMs of Validated Peptides of Validated Proteins
Validated PSMs of Validated Peptides
Validated PSMs
Confidently localized PTMs of Validated PSMs of Validated Peptides of Validated Proteins

Exported identification results are in Progenesis LC-MS compatible format as TXT.

Inclusion/Exclusion list

Exports an inclusion list of validated hits.

Inclusion list may be filtered according to peptide types:

Miscleaved Peptides
Reactive Peptides
Degenerated Peptides.

and also according to protein inferences:

Related Proteins
Related and Unrelated Proteins
Unrelated Proteins.

Finally, a retention time window may be established as a final filter.

Exported results can be in Thermo, ABI, Bruker or MassLynx formats.

Identification Features Reports

PSM Report

Column	Description
Protein(s)	Protein(s) to which the peptide can be attached
Sequence	Sequence of the peptide
AAs Before	The amino acids before the sequence
AAs After	The amino acids after the sequence
Position	Position of the peptide in the protein sequence(s).
Modified Sequence	The amino acids sequence annotated with variable modifications.
Variable Modifications	The variable modifications
Fixed Modifications	The fixed modifications.
Spectrum File	The spectrum file.
Spectrum Title	The title of the spectrum.
Spectrum Scan Number	The spectrum scan number.
RT	Retention time
m/z	Measured m/z
Measured Charge	The charge as given in the spectrum file.
Identification Charge	The charge as inferred by the search engine.
Theoretical Mass	The theoretical mass of the peptide.
Isotope Number	The isotope number targetted by the instrument.
Precursor m/z Error [ppm]	The precursor m/z matching error.
Localization Confidence	The confidence in variable PTM localization.
probabilistic PTM score	The probabilistic score (e.g. A-score or PhosphoRS) used for variable PTM localization.
D-score	D-score for variable PTM localization
Confidence	Confidence in percent associated to the retained PSM.
Validation	Indicates the validation level of the protein group.

PSM Report with non-validated matches

Same columns as the original PSM report but also includes non-validated matches.

PSM Phosphorylation Report

Column	Description
Protein(s)	Protein(s) to which the peptide can be attached
Sequence	Sequence of the peptide
Variable Modifications	The variable modifications
Fixed Modifications	The fixed modifications.
Spectrum File	The spectrum file.
Spectrum Title	The title of the spectrum.
Spectrum Scan Number	The spectrum scan number.
RT	Retention time
m/z	Measured m/z
Measured Charge	The charge as given in the spectrum file.
Identification Charge	The charge as inferred by the search engine.
Theoretical Mass	The theoretical mass of the peptide.
Isotope Number	The isotope number targetted by the instrument.
Precursor m/z Error [ppm]	The precursor m/z matching error.
Localization Confidence	The confidence in variable PTM localization.
probabilistic PTM score	The probabilistic score (e.g. A-score or PhosphoRS) used for variable PTM localization.
D-score	D-score for variable PTM localization
Confident Phosphosites	List of the sites where a phosphorylation was confidently localized.
#Confident Phosphosites	Number of confidently localized phosphorylations.
Ambiguous Phosphosites	List of the sites where a phosphorylation was ambiguously localized.
#Ambiguous Phosphosites	Number of ambiguously localized phosphorylations.
Confidence [%]	Confidence in percent associated to the retained PSM.
Validation	Indicates the validation level of the protein group.

Extended PSM Report

Same as the ordinary PSM report but adds a Decoy column and lacks the Confidence column.

Extra column	Description
Decoy	Indicates whether the peptide is a decoy (1: yes, 0: no).

Peptide Report

Column	Description
Protein(s):	Protein(s) to which this peptide can be attached.
Protein Group(s)	List of identified protein groups this peptide can map to with associated validation level.
#Validated Protein Group(s)	Indicates the number of protein groups this peptide maps to.
Unique Group	Indicates whether the peptide maps to a unique protein group.
Sequence	Sequence of the peptide.
Modified Sequence	The peptide sequence annotated with variable modifications.
Position	Position of the peptide in the protein sequence(s).
AAs Before	The amino acids before the sequence.
AAs After	The amino acids after the sequence.
Variable Modifications	The variable modifications.
Fixed Modifications	The fixed modifications.
Localization Confidence	The confidence in PTMs localization.
#Validated PSMs	Number of validated PSMs.
#PSMs	Number of PSMs.
Confidence [%]	Confidence in percent associated to the peptide.
Validation	Indicates the validation level of the peptide.

Peptide Report with non-validated matches

Same columns as the original Peptide report but also includes non-validated matches.

Peptide Phosphorylation Report

Column	Description
Protein(s):	Protein(s) to which this peptide can be attached.
Protein Group(s)	List of identified protein groups this peptide can map to with associated validation level.
#Validated Protein Group(s)	Indicates the number of protein groups this peptide maps to.
Unique Group	Indicates whether the peptide maps to a unique protein group.
Sequence	Sequence of the peptide.
Modified Sequence	The peptide sequence annotated with variable modifications.
AAs Before	The amino acids before the sequence.
AAs After	The amino acids after the sequence.
Variable Modifications	The variable modifications.
Fixed Modifications	The fixed modifications.
Localization Confidence	The confidence in PTMs localization.
Confident Phosphosites	List of the sites where a phosphorylation was confidently localized.
#Confident Phosphosites	Number of confidently localized phosphorylations.
Ambiguous Phosphosites	List of the sites where a phosphorylation was ambiguously localized.
#Ambiguous Phosphosites	Number of ambiguously localized phosphorylations.
#Validated PSMs	Number of validated PSMs.
#PSMs	Number of PSMs.
Confidence [%]	Confidence in percent associated to the peptide.
Validation	Indicates the validation level of the peptide.

Protein Report

Column	Description
Main Accession	Main accession of the protein group.
Description	Description of the protein designed by the main accession.
Gene Name	The gene names of the Ensembl gene ID associated to the main accession.
Chromosome	The chromosome of the Ensembl gene ID associated to the main accession.
MW (kDa)	Molecular Weight.
Possible Coverage (%)	Possible sequence coverage in percent of the protein designed by the main accession according to the search settings.
Coverage (%)	Sequence coverage in percent of the protein designed by the main accession.
Spectrum Counting	The selected spectrum counting metric.
Confidently Localized Modification Sites	List of the sites where a variable modification was confidently localized.
# Confidently Localized Modification Sites	Number of sites where a variable modification was confidently localized.
Ambiguously Localized Modification Sites	List of the sites where ambiguously localized variable modification could possibly be located.
#Ambiguously Localized Modification Sites	Number of ambiguously localized modifications.
Protein Inference	Protein Inference status of the protein group.
Secondary Accessions	Other accessions in the protein group (alphabetical order).
Protein Group	The complete protein group (alphabetical order).
#Validated Peptides	Number of validated peptides.
#Peptides	Total number of peptides.
#Unique	Total number of peptides unique to this protein group.
#Validated Unique	Total number of peptides unique to this protein group.
#Validated PSMs	Number of validated PSMs.
#PSMs	Number of PSMs.
Confidence [%]	Confidence in percent associated to the protein group.
Validation	Indicates the validation level of the protein group.

Protein Report with non-validated matches

Same columns as the original Protein report but also includes non-validated matches.

Protein Phosphorylation Report

Column	Description
Main Accession	Main accession of the protein group.
Description	Description of the protein designed by the main accession.
Gene Name	The gene names of the Ensembl gene ID associated to the main accession.
Chromosome	The chromosome of the Ensembl gene ID associated to the main accession.
MW (kDa)	Molecular Weight.
Possible Coverage (%)	Possible sequence coverage in percent of the protein designed by the main accession according to the search settings.
Coverage (%)	Sequence coverage in percent of the protein designed by the main accession.
Spectrum Counting NSAF	Raw Normalized Spectrum Abundance Factor (NSAF).
Confident Phosphosites	List of the sites where a phosphorylation was confidently localized.
#Confident Phosphosites	Number of sites where a phosphorylation was confidently localized.
Ambiguous Phosphosites	List of the sites where a phosphorylation was ambiguously localized.
#Ambiguous Phosphosites	Number of sites where a phosphorylation was ambiguously localized.
Protein Inference	Protein Inference status of the protein group.
Secondary Accessions	Other accessions in the protein group (alphabetical order).
Protein Group	The complete protein group (alphabetical order).
#Validated Peptides	Number of validated peptides.
#Peptides	Total number of peptides.
#Unique	Total number of peptides unique to this protein group.
#Validated Unique	Total number of peptides unique to this protein group.
#Validated PSMs	Number of validated PSMs.
#PSMs	Number of PSMs.
Confidence [%]	Confidence in percent associated to the protein group.
Validation	Indicates the validation level of the protein group.

Certificate of Analysis

Presents a summary of the most important statistics of the search, grouped by:

Project Details:
- PeptideShaker Version, Date, Experiment, Sample, Replicate Number, Identification Algorithms.
Database Search Parameters:
- Precursor Tolerance Unit, Precursor Ion m/z Tolerance, Fragment Ion Tolerance Unit, Fragment Ion m/z Tolerance, Cleavage, Enzyme, Missed Cleavages, Specificity, Database, Forward Ion, Rewind Ion, Fixed Modifications, Variable Modifications, Refinement Variable Modifications, Refinement Fixed Modifications.
Input Filters:
- Minimal Peptide Length, Maximal Peptide Length, Precursor m/z Tolerance, Precursor m/z Tolerance Unit, Unrecognized Modifications Discarded.
Validation Summary:
- Proteins:
  
  #Validated, Total Possible TP, FDR Limit [%], FNR Limit [%], Confidence Limit, PEP Limit [%], Confidence Accuracy [%].
- Peptides:
  
  #Validated, Total Possible TP, FDR Limit [%], FNR Limit [%], Confidence Limit [%], PEP Limit [%], Confidence Accuracy [%].
- PSMs:
  
  #Validated PSM, Total Possible TP, FDR Limit [%]: PSMs, Confidence Limit [%], PEP Limit [%], Confidence Accuracy [%].
PTM Scoring Settings:
- Probabilistic Score, Accounting for Neutral Losses, Threshold.
Spectrum Counting Parameters:
- Method, Validated Matches Only
Annotation Settings:
- Intensity Limit, Automatic Annotation, Selected Ions, Neutral Losses, Neutral Losses Sequence Dependence, Fragment Ion m/z Tolerance.

Hierachical Report

Column	Description
Main Accession	Main accession of the protein group.
Description	Description of the protein designed by the main accession.
MW (kDa)	Molecular Weight.
Possible Coverage (%)	Possible sequence coverage in percent of the protein designed by the main accession according to the search settings.
Coverage (%)	Sequence coverage in percent of the protein designed by the main accession.
Spectrum Counting NSAF	Normalized Spectrum Abundance Factor (NSAF)
Confidently Localized Modification Sites	List of the sites where a variable modification was confidently localized.
# Confidently Localized Modification Sites	Number of sites where a variable modification was confidently localized.
Ambiguously Localized Modification Sites	List of the sites where ambiguously localized variable modification could possibly be located.
#Ambiguously Localized Modification Sites	Number of ambiguously localized modifications.
Protein Inference	Protein Inference status of the protein group.
Secondary Accessions	Other accessions in the protein group (alphabetical order).
Protein Group	The complete protein group (alphabetical order).
#Validated Peptides	Number of validated peptides.
#Peptides	Total number of peptides.
#Unique	Total number of peptides unique to this protein group.
#Validated PSMs	Number of validated PSMs
#PSMs	Number of PSMs
Confidence	Confidence in percent associated to the protein group.
Decoy	Indicates whether the protein group is a decoy (1: yes, 0: no).
Validation	Indicates the validation level of the protein group.

Citation

To cite the underlying tools (PeptideShaker and SearchGUI) please refer to the list of papers at http://compomics.github.io

If you use this tool in Galaxy, please cite Chilton J, Ira Cooke, Bjoern Gruening et al.