MS-GFDB

MS-GFDB has been renamed to MS-GF+ . MS-GF+ has all the functionalities provided by MS-GFDB.

Differences of MS-GF+ compared to MS-GFDB

  • Input
    • Supports mzML in addition to mzXML, mgf, ms2, pkl and _dta.txt
    • "-t PrecursorMassTolerance" became an optional parameter (default 20ppm)
    • "-c13 0/1/2" was replaced to "-ti IsotopeErrorRange". 
      • IsotopeErrorRange: MinIsotopeError,MaxIsotopeError (both are inclusive)
      • -c13 x == -ti 0,x
    • "-nnet" was replaced to "-ntt".
      • -nnet 0 == -ntt 2, -nnet 1 == -ntt 1, -nnet 2 == -ntt 0
    • ModificationFile format change
      • The name of the modification should match the PSI-MS name (accessible from http://www.unimod.org)
      • CompositionStr can take Br, Cl, Fe, Se in addition to C, H, N, O, S, and P.
      • The sequence of the atoms can be arbitrary. 
        • Previously C2H2O was valid but OH2C2 was invalid.
        • Now, both are valid.
    • "-uniformAAProb 0/1" was deleted.
    • "-addFeatures 0/1" was added. "-addFeatures 1" will output the following extra features for each PSM (will be useful to downstream tools like Percolator or IDPicker):
      • MS2IonCurrent: Summed intensity of all product ions
      • ExplainedIonCurrentRatio: Summed intensity of all matched product ions (e.g. b, b-H2O, y, etc.) divided by MS2IonCurrent
      • NTermIonCurrentRatio: Summed intensity of all matched prefix ions (e.g. b, b-H2O, etc.) divided by MS2IonCurrent
      • CTermIonCurrentRatio: Summed intensity of all matched suffix ions (e.g. y, y-H2O, etc.) divided by MS2IonCurrent
    • "-showQValue 0/1" was added.
    • "-showDecoy 0/1" was added.
  • Output
    • Output format is the HUPO PSI mzIdentML version 1.1 (*.mzid).*** See http://www.psidev.info/mzidentml for details.
      • Decoy protein prefix was changed to "XXX" ("REV" previously).
    • Provides a converter from mzIdentML to tsv (the resulting tsv file will be similar to the MS-GFDB output file).
      • The converter is included in the MSGFPlus.jar file.
      • Can be run by "java -Xmx2000M edu.ucsd.msjava.ui.MzIDToTsv".
    • Difference between the MS-GFDB output and the MS-GF+ tsv output
      • SpecID (native spectrum ID) is reported instead of SpecIndex
      • IsotopeError is reported.
      • When a peptide matches to multiple proteins, all protein accessions will be reported.
      • SpecProb was renamed to SpecEValue
      • EValue (database-level E-value) is reported instead of PValue (database-level P-value).
      • FDR and PepFDR were renamed to QValue and  PepQValue, respectively.

MS-GFDB

Usage: java -Xmx2000M -jar MSGFDB.jar
	-s SpectrumFile (*.mzXML, *.mzML, *.mgf, *.ms2, *.pkl or *_dta.txt)
	-d DatabaseFile (*.fasta or .fa)
	-t ParentMassTolerance (e.g. 2.5Da, 30ppm, or 0.5Da,2.5Da)
	   Use comma to set asymmetric values. E.g. "-t 0.5Da,2.5Da" will set 0.5Da to the left (expMass<theoMass) and 2.5Da to the right (expMass>theoMass).
	[-o outputFileName] (Default: stdout)
	[-thread NumOfThreads] (Number of concurrent threads to be executed, Default: Number of available cores)
	[-tda 0/1] (0: don't search decoy database (default), 1: search decoy database to compute FDR)
	[-m FragmentationMethodID] (0: as written in the spectrum or CID if no info (Default), 1: CID, 2: ETD, 3: HCD, 4: Merge spectra from the same precursor)
	[-inst InstrumentID] (0: Low-res LCQ/LTQ (Default for CID and ETD), 1: High-res LTQ (Default for HCD), 2: TOF)
	[-e EnzymeID] (0: No enzyme, 1: Trypsin (Default), 2: Chymotrypsin, 3: Lys-C, 4: Lys-N, 5: Glu-C, 6: Arg-C, 7: Asp-N, 8: aLP, 9: Endogenous peptides)
	[-c13 0/1/2] (Number of allowed C13, Default: 1)
	[-nnet 0/1/2] (Number of allowed non-enzymatic termini, Default: 1)
	[-mod ModificationFileName] (Modification file, Default: standard amino acids with fixed C+57)
	[-minLength MinPepLength] (Minimum peptide length to consider, Default: 6)
	[-maxLength MaxPepLength] (Maximum peptide length to consider, Default: 40)
	[-minCharge MinPrecursorCharge] (Minimum precursor charge to consider if not specified in the spectrum file, Default: 2)
	[-maxCharge MaxPrecursorCharge] (Maximum precursor charge to consider if not specified in the spectrum file, Default: 3)
	[-n NumMatchesPerSpec] (Number of matches per spectrum to be reported, Default: 1)
	[-uniformAAProb 0/1] (0: use amino acid probabilities computed from the input database (default), 1: use probability 0.05 for all amino acids)
Parameters:
  • -s SpectrumFile (*.mzXML, *.mzML, *.mgf, *.ms2, *.pkl or *_dta.txt) - Required
    • Spectrum file name. Currently, MS-GFDB supports the following file formats: mzXML, mzML, mgf, ms2, pkl and _dta.txt.
  • -d DatabaseFile (*.fasta or *.fa) - Required
    • Path to the protein database file. If the database file does not have auxiliary index files (*.canno, *.cnlcp, *.csarr, and *.cseq), MS-GFDB will create them.
    • When "-tda 1" option is used, the database must contain only target protein sequences.
If multiple MS-GFDB processes access the same database file, it is strongly recommended to index the database prior to the database search by running BuildSA (see below).
  • -t ParentMassTolerance - Required
    • Parent mass tolerance in Da. or ppm. There must be no space between the number and the unit. E.g. 2.5Da, 30ppm
    • To set asymmetric tolerances, use comma to separate left (experimental mass < theoretical mass) or right (experimental mass > theoretical mass) tolerances. E.g. 0.5Da,2.5Da
  • -o OutputFile (Default: stdout)
    • Filename where the output will be written.
    • The output will be printed to standard out by default.
  • -thread NumOfThreads (Number of concurrent threads to be executed, Default: Number of available cores)
    • Number of concurrent threads to be executed together.
    • Default value is the number of available logical cores (e.g. 8 for quad-core processor with hyper-threading support).
  • -tda 0/1 (0: don't search decoy database (default), 1: search decoy database to compute FDR)
    • Indicates whether to search the decoy database or not.
    • If 0, the decoy database is not searched and FDRs are theoretically derived from P-values (EFDR).
    • If 1, FDRs are computed based on the target-decoy approach (i.e. reversed database is appended to the target database and MS-GFDB searches the combined database)
      • FDR(t) = #(DecoyPSMs with score equal or above t) / #(TargetPSMs with score equal or above t).
      • PSM: Peptide-Spectrum Match
      • -log(SpecProb) is used as the score to compute FDR.
If -tda 1 is specified, MS-GFDB automatically creates a combined target/reversed database file (DBFileName.revConcat.fasta). Thus, when specifying "-d" parameter, DatabaseFile must contain only target proteins.
  • -m FragmentationMethodID (0: as written in the spectrum or CID if no info (Default), 1: CID, 2: ETD, 3: HCD, 4: Merge spectra from the same precursor)
    • Fragmentation method identifier (used to determine the scoring model).
    • If the identifier is 0 and fragmentation method is written in the spectrum file (e.g. activationMethod field in mzXML files), MS-GFDB will recognize the fragmentation method and use a relevant scoring model.
    • If the identifier is 0 and there is no fragmentation method information in the spectrum (e.g. mgf files), CID model will be used by default.
    • If the identifier is non-zero and the spectrum has fragmentation method information, only the spectra that match with the identifier will be processed.
    • If the identifier is non-zero and the spectrum has no fragmentation method information, MS-GFDB will process all spectra assuming the specified fragmentation method.
    • If the identifier is 4, MS/MS spectra from the same precursor ion (e.g. CID/ETD pairs, CID/HCD/ETD triplets) will be merged and the "merged" spectrum will be used for searching instead of individual spectra. See Kim et al., MCP 2010 for details.
  • -inst InstrumentID (0: Low-res LCQ/LTQ (Default for CID and ETD), 1: TOF , 2: High-res LTQ (Default for HCD))
    • Identifier of the instrument to generate MS/MS spectra (used to determine the scoring model).
    • For "hybrid" spectra with high-precision MS1 and low-precision MS2, use 0.
    • For usual low-precision instruments (e.g. Thermo LTQ), use 0.
    • For TOF instruments, use 1.
    • If MS/MS fragment ion peaks are of high-precision (e.g. tolerance = 10ppm), use 2.
  • -e EnzymeID (Default: 1)
    • Enzyme identifier. Trypsin (1) will be used by default.
    • 0: No enzyme, 1: Trypsin (default), 2: Chymotrypsin, 3: Lys-C, 4: Lys-N, 5: Glu-C, 6: Arg-C, 7: Asp-N, 8: alphaLP, 9: Endogenous peptides
  • -c13 0/1/2 (Number of allowed isotope errors, Default: 1)
    • Instruments often choose 2nd or 3rd isotope peak instead of mono-isotope peak from MS1 spectrum.
    • If this value is non-zero, expPeptideMass-1.00335 (i.e. mass(13C)-mass(12C)) and expPeptideMass-2.00671 (i.e. 2*(mass(C13)-mass(C12)) (only if -c13 2) will be considered along with expPeptideMass.
    • If accurate precursor ion mass is available (e.g. LTQ-Orbitrap), it is better to set a narrow parent mass tolerance and non-zero -c13 value (e.g. -t 30ppm -c13 1) than to set a wide tolerance (e.g. -t 0.5Da,2.5Da).
    • If the parent mass tolerance is equal to or larger than 0.5Da or 500ppm, this parameter will be ignored.
  • -nnet 0/1/2 (Number of allowed non-enzymatic termini, Default: 1)
    • This parameter is used to determine the enzyme cleavage rule.
    • Specifies the maximum number of peptide termini that are not cleaved by the enzyme.
    • For example, for trypsin, K.ACDEFGHR.C, G.ACDEFGHR.C, K.ACDEFGHI.C and G.ACDEFGHR.C have 0, 1, 1 and 2 non-enzymatic termini, accordingly.
    • By default, -nnet 1 will be used. Using -nnet 0 (or 2) will make the search significantly faster (slower).
  • -mod ModificationFile (Default: standard amino acids with fixed C+57)]
    • Modification file name. ModificationFile contains the modifications to be considered in the search.
    • If -mod option is not specified, standard amino acids with fixed Carboamidomethylation C will be used.
    • See an example modification file .
  • -minLength MinPepLength (Default: 6)
    • Minimum length of the peptide to be considered.
  • -maxLength MaxPepLength (Default: 40)
    • Maximum length of the peptide to be considered.
  • -minCharge MinPrecursorCharge (Default: 2)
    • Minimum precursor charge to consider. This parameter is used only for spectra with no charge.
  • -maxCharge MinPrecursorCharge (Default: 3)
    • Maximum precursor charge to consider. This parameter is used only for spectra with no charge.
  • -n NumMatchesPerSpec (Default: 1)
    • Number of peptide matches per spectrum to report.
    • Expected false discovery rates (EFDRs) will be reported only when this value is 1.
  • -uniformAAProb 0/1 (Default: 0)
    • If 0, compute amino acid frequencies from the input database and use them as amino acid probabilities.
    • If 1, use uniform amino acid probability (preferable when the database size is small).
MS-GFDB output

MS-GFDB outputs a tab-delimited file with the following columns: #SpecFile, Scan#, FragMethod, Precursor, PMError, Charge, Peptide, Protein, DeNovoScore, MSGFScore, SpecProb, P-value, EFDR.

  • SpecFile: spectrum file name
  • SpecIndex: spectrum index (1-based) in the file. The first spectrum has index 1, the second has index 2, and so on. For mzXML files this value is same as the scan number.
  • Scan#: scan number of the spectrum. If the scan number is not available, the value will be -1.
  • FragMethod: fragmentation method used to generate the spectrum (e.g. CID, ETD, etc.). When spectra from the same precursor are merged, fragmentation methods of merged spectra will be shown as a form "FragMethod1/FragMethod2/..." (e.g. CID/ETD, CID/HCD/ETD).
  • Precursor: precursor mass in m/z or ppm
  • Charge: precursor ion charge
  • Peptide: peptide sequence with neighboring amino acids
  • Protein: protein name
  • DeNovoScore: the score of the optimal scoring peptide (not necessary in the database)
  • MSGFScore: MS-GF raw score of the peptide-spectrum match (MSGFScore <= DeNovoScore)
  • SpecProb: spectral probability (spectrum level p-value) of the peptide-spectrum match
  • P-value: database level p-value (probability that a random PSM have an equal or better score against a random database of the same size)
  • EFDR or FDR: false discovery rate
    • If "-tda 1" is specified, FDRs are estimated using the target-decoy approach using the spectral probability (SpecProb) as the score (the lower, the better).
    • Otherwise, FDRs are estimated using P-values without searching the decoy database (EFDR). See Gupta et al., JASMS 2011 for details.
    • MS-GFDB reports EFDR only when it is configured to report 1 peptide match per spectrum (i.e. -n 1).
    • EFDR accurately estimates FDR when the parent mass tolerance is equal or larger than 0.5.
    • EFDR conservatively estimates FDR when the parent mass tolerance is small.
      • E.g. When parent mass tolerance is 30ppm, at EFDR 1% threshold, one identifies approximately 7% less peptide-spectrum matches (PSMs) compared to the case when the target-decoy approach is used to estimate the FDR.
  • PepFDR
    • Peptide-level FDR estimated using the target-decoy approach.
    • Reported only if "-tda 1" is specified.
    • If multiple spectra are matched to the same peptide, only the best scoring PSM (lowest SpecProb) is retained. After that, PepFDR is calculated as #DecoyPSMs>s / #TargetPSMs>s among the retained PSMs. This approximates the FDR of the set of unique peptides. In the MS-GFDB output, the same PepFDR value is given to all PSMs sharing the peptide. So, even a low-quality PSM may get a low PepFDR value (if it has a high-quality "sibling" PSM sharing the peptide). Note that this should not be used to count the number of identified PSMs.
MS-GFDB output example

#SpecFile SpecIndex Scan# FragMethod Precursor PMError(ppm) Charge Peptide Protein DeNovoScore MSGFScore SpecProb P-value FDR PepFDR
090121_NM_Trypsin_20.mzXML 2838 2838 CID 964.7707 1.5199227 3 K.TIQNSSVSPTSSSSSSSSTGETQTQSSSR.L IPI:IPI00002349.2|SWISS-PROT:Q7Z417|TREMBL:A1L3A7|ENSEMBL:ENSP00000225388|REFSEQ:NP_065823|H-INV:HIT000001036|VEGA:OTTHUMP00000181037 Tax_Id=9606 Gene_Symbol=NUFIP2 Nuclear fragile X mental retardation-interacting protein 2 190 181 9.380133E-30 2.9333857E-22 0.0 0.0
090121_NM_Trypsin_20.mzXML 3671 3671 ETD 1113.4758 0.6583758 2 R.VGPADDGPAPSGEEEGEGGGEAGGK.E IPI:IPI00016725.2|SWISS-PROT:Q9UKN8|TREMBL:B3KNH2;Q05CN7|ENSEMBL:ENSP00000361219|REFSEQ:NP_036336|H-INV:HIT000071196|VEGA:OTTHUMP00000022434 Tax_Id=9606 Gene_Symbol=GTF3C4 General transcription factor 3C polypeptide 4 162 158 1.9912463E-28 6.0892146E-21 0.0 0.0
090121_NM_Trypsin_20.mzXML 3031 3031 ETD 651.64874 1.7510794 3 K.GAAAAAAASGAAGGGGGGAGAGAPGGGR.L IPI:IPI00644073.1|VEGA:OTTHUMP00000038687 Tax_Id=9606 Gene_Symbol=INTS3 18 kDa protein 214 202 6.7318633E-28 2.093763E-20 0.0 0.0
090121_NM_Trypsin_20.mzXML 19088 19088 CID 1199.0916 10.392676 2 K.VNFSPPGDTNSLFPGTWYLER.V IPI:IPI00945760.1|TREMBL:B7Z784;B7Z7M8;B7Z8R3|REFSEQ:NP_001159579 Tax_Id=9606 Gene_Symbol=HMGCS2 hydroxymethylglutaryl-CoA synthase, mitochondrial isoform 2 precursor 243 243 2.9611275E-27 8.838129E-20 0.0 0.0
090121_NM_Trypsin_20.mzXML 3030 3030 CID/ETD 651.64874 1.7510794 3 K.GAAAAAAASGAAGGGGGGAGAGAPGGGR.L IPI:IPI00644073.1|VEGA:OTTHUMP00000038687 Tax_Id=9606 Gene_Symbol=INTS3 18 kDa protein 389 389 7.508096E-33 2.335189E-25 0.0 0.0

BuildSA

Index a protein database for fast searching. 

Usage: java -cp MSGFDB.jar msdbsearch.BuildSA 
	-d DatabaseFile (*.fasta or *.fa)
	[-tda 0/1/2] (0: target only, 1: target-decoy database only, 2: both)

Parameters:

  • -d DbPath
    • Name of a protein database (*.fasta or *.fa) 
    • Database file must ends with ".fasta" or ".fa".
  • -tda 0/1/2
    • If 0, only "DatabaseFile" will be indexed.
    • If 1, a new database file (*.revConcat.fasta) will be generated by appending reversed proteins. This forward-reverse database will be indexed.
    • If 2, both the original database and the forward-reverse database file will be indexed.

BuildSA creates a suffix array of the protein database. For a input database file DBFileName.fasta, BuildSA will generate 4 auxiliary files (DbFileName.canno, DBFileName.cnlcp, DBFileName.csarr, DBFileName.cseq). It needs to be executed only once per each database file.

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  1. May 17, 2012

    Anonymous says:

    Hello, Is it possible to perform the db-search on multiple datasets, i.e. multi...

    Hello,

    Is it possible to perform the db-search on multiple datasets, i.e. multiple data files coming from multiple LC-MS/MS runs?

    Thanks,

    G.E.

  2. Jun 28, 2012

    Anonymous says:

    Can I search for PTMs using negative monoisotopic mass in the Mods file? Thanks...

    Can I search for PTMs using negative monoisotopic mass in the Mods file?

    Thanks,

    Fernanda

  3. Aug 27, 2012

    Anonymous says:

    mzML appears to be not supported in v7780 MSGFDB. Not a big thing, but for docum...

    mzML appears to be not supported in v7780 MSGFDB. Not a big thing, but for documentation purpose, I think it needs to be updated.

  4. Oct 15, 2012

    Anonymous says:

    Is it possible to modify the program so that thermolysin can be used as a cleava...

    Is it possible to modify the program so that thermolysin can be used as a cleavage enzyme?

  5. Nov 28, 2013

    Anonymous says:

    Nice info.. I want to know that is it possible db-search on multiple datasets.....

    Nice info..

    I want to know that is it possible db-search on multiple datasets....

    livingfilms

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