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EMBOSS: pepdigest

pepdigest

Wiki

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Function

Reports on protein proteolytic enzyme or reagent cleavage sites

Description

This programs allows you to input one or more protein sequences and to specify one proteolytic agent from a list, which might be a proteolytic enzyme or other reagent. It will then write a report file containing the positions where the agent cuts, together with the peptides produced.

Algorithm

By default pepdigest uses average molecular weights. The -mono option will use monoisotopic weights.

Usage

Here is a sample session with pepdigest


% pepdigest 
Reports on protein proteolytic enzyme or reagent cleavage sites
Input protein sequence(s): tsw:opsd_human
Enzymes and Reagents
         1 : Trypsin
         2 : Lys-C
         3 : Arg-C
         4 : Asp-N
         5 : V8-bicarb
         6 : V8-phosph
         7 : Chymotrypsin
         8 : CNBr
Select number [1]: 
Use monoisotopic weights [N]: 
Output report [opsd_human.pepdigest]:

Go to the input files for this example
Go to the output files for this example

Command line arguments

Reports on protein proteolytic enzyme or reagent cleavage sites
Version: EMBOSS:6.4.0.0

   Standard (Mandatory) qualifiers:
  [-seqall]            seqall     Protein sequence(s) filename and optional
                                  format, or reference (input USA)
   -menu               menu       [1] Select number (Values: 1 (Trypsin); 2
                                  (Lys-C); 3 (Arg-C); 4 (Asp-N); 5
                                  (V8-bicarb); 6 (V8-phosph); 7
                                  (Chymotrypsin); 8 (CNBr))
   -mono               boolean    [N] Use monoisotopic weights
  [-outfile]           report     [*.pepdigest] Output report file name
                                  (default -rformat seqtable)

   Additional (Optional) qualifiers: (none)
   Advanced (Unprompted) qualifiers:
   -mwdata             datafile   [Emolwt.dat] Molecular weight data for amino
                                  acids
   -unfavoured         boolean    Trypsin will not normally cut after 'KR' if
                                  they are followed by any of 'KRIFLP'. Lys-C
                                  will not normally cut after 'K' if it is
                                  followed by 'P'. Arg-C will not normally cut
                                  after 'R' if it is followed by 'P'.
                                  V8-bicarb will not normally cut after 'E' if
                                  it is followed by any of 'KREP'. V8-phosph
                                  will not normally cut after 'DE' if they are
                                  followed by 'P'. Chymotrypsin will not
                                  normally cut after 'FYWLM' if they are
                                  followed by 'P'. Specifying unfavoured shows
                                  these unfavoured cuts as well as the
                                  favoured ones.
   -ragging            boolean    Allows semi-specific and non-specific
                                  digestion. This option is particularly
                                  useful for generating lists of peptide
                                  sequences for protein identification using
                                  mass-spectrometry.
   -termini            menu       [1] Select number (Values: 1 (none); 2
                                  (nterm); 3 (cterm); 4 (nterm OR cterm))
   -overlap            boolean    Used for partial digestion. Shows all cuts
                                  from favoured cut sites plus 1..3, 2..4,
                                  3..5 etc but not (e.g.) 2..5. Overlaps are
                                  therefore fragments with exactly one
                                  potential cut site within it.
   -allpartials        boolean    As for overlap but fragments containing more
                                  than one potential cut site are included.

   Associated qualifiers:

   "-seqall" associated qualifiers
   -sbegin1            integer    Start of each sequence to be used
   -send1              integer    End of each sequence to be used
   -sreverse1          boolean    Reverse (if DNA)
   -sask1              boolean    Ask for begin/end/reverse
   -snucleotide1       boolean    Sequence is nucleotide
   -sprotein1          boolean    Sequence is protein
   -slower1            boolean    Make lower case
   -supper1            boolean    Make upper case
   -sformat1           string     Input sequence format
   -sdbname1           string     Database name
   -sid1               string     Entryname
   -ufo1               string     UFO features
   -fformat1           string     Features format
   -fopenfile1         string     Features file name

   "-outfile" associated qualifiers
   -rformat2           string     Report format
   -rname2             string     Base file name
   -rextension2        string     File name extension
   -rdirectory2        string     Output directory
   -raccshow2          boolean    Show accession number in the report
   -rdesshow2          boolean    Show description in the report
   -rscoreshow2        boolean    Show the score in the report
   -rstrandshow2       boolean    Show the nucleotide strand in the report
   -rusashow2          boolean    Show the full USA in the report
   -rmaxall2           integer    Maximum total hits to report
   -rmaxseq2           integer    Maximum hits to report for one sequence

   General qualifiers:
   -auto               boolean    Turn off prompts
   -stdout             boolean    Write first file to standard output
   -filter             boolean    Read first file from standard input, write
                                  first file to standard output
   -options            boolean    Prompt for standard and additional values
   -debug              boolean    Write debug output to program.dbg
   -verbose            boolean    Report some/full command line options
   -help               boolean    Report command line options and exit. More
                                  information on associated and general
                                  qualifiers can be found with -help -verbose
   -warning            boolean    Report warnings
   -error              boolean    Report errors
   -fatal              boolean    Report fatal errors
   -die                boolean    Report dying program messages
   -version            boolean    Report version number and exit

Qualifier

Type

Description

Allowed values

Default

Standard (Mandatory) qualifiers

[-seqall]
(Parameter 1)

seqall

Protein sequence(s) filename and optional format, or reference (input USA)

Readable sequence(s)

Required

-menu

list

Select number

1	(Trypsin)
2	(Lys-C)
3	(Arg-C)
4	(Asp-N)
5	(V8-bicarb)
6	(V8-phosph)
7	(Chymotrypsin)
8	(CNBr)

-mono

boolean

Use monoisotopic weights

Boolean value Yes/No

[-outfile]
(Parameter 2)

report

Output report file name

(default -rformat seqtable)

<*>.pepdigest

Additional (Optional) qualifiers

(none)

Advanced (Unprompted) qualifiers

-mwdata

datafile

Molecular weight data for amino acids

Data file

Emolwt.dat

-unfavoured

boolean

Trypsin will not normally cut after 'KR' if they are followed by any of 'KRIFLP'. Lys-C will not normally cut after 'K' if it is followed by 'P'. Arg-C will not normally cut after 'R' if it is followed by 'P'. V8-bicarb will not normally cut after 'E' if it is followed by any of 'KREP'. V8-phosph will not normally cut after 'DE' if they are followed by 'P'. Chymotrypsin will not normally cut after 'FYWLM' if they are followed by 'P'. Specifying unfavoured shows these unfavoured cuts as well as the favoured ones.

Boolean value Yes/No

-ragging

boolean

Allows semi-specific and non-specific digestion. This option is particularly useful for generating lists of peptide sequences for protein identification using mass-spectrometry.

Boolean value Yes/No

-termini

list

Select number

1	(none)
2	(nterm)
3	(cterm)
4	(nterm OR cterm)

-overlap

boolean

Used for partial digestion. Shows all cuts from favoured cut sites plus 1..3, 2..4, 3..5 etc but not (e.g.) 2..5. Overlaps are therefore fragments with exactly one potential cut site within it.

Boolean value Yes/No

-allpartials

boolean

As for overlap but fragments containing more than one potential cut site are included.

Boolean value Yes/No

Associated qualifiers

"-seqall" associated seqall qualifiers

-sbegin1
-sbegin_seqall

integer

Start of each sequence to be used

Any integer value

-send1
-send_seqall

integer

End of each sequence to be used

Any integer value

-sreverse1
-sreverse_seqall

boolean

Reverse (if DNA)

Boolean value Yes/No

-sask1
-sask_seqall

boolean

Ask for begin/end/reverse

Boolean value Yes/No

-snucleotide1
-snucleotide_seqall

boolean

Sequence is nucleotide

Boolean value Yes/No

-sprotein1
-sprotein_seqall

boolean

Sequence is protein

Boolean value Yes/No

-slower1
-slower_seqall

boolean

Make lower case

Boolean value Yes/No

-supper1
-supper_seqall

boolean

Make upper case

Boolean value Yes/No

-sformat1
-sformat_seqall

string

Input sequence format

Any string

-sdbname1
-sdbname_seqall

string

Database name

Any string

-sid1
-sid_seqall

string

Entryname

Any string

-ufo1
-ufo_seqall

string

UFO features

Any string

-fformat1
-fformat_seqall

string

Features format

Any string

-fopenfile1
-fopenfile_seqall

string

Features file name

Any string

"-outfile" associated report qualifiers

-rformat2
-rformat_outfile

string

Report format

Any string

seqtable

-rname2
-rname_outfile

string

Base file name

Any string

-rextension2
-rextension_outfile

string

File name extension

Any string

-rdirectory2
-rdirectory_outfile

string

Output directory

Any string

-raccshow2
-raccshow_outfile

boolean

Show accession number in the report

Boolean value Yes/No

-rdesshow2
-rdesshow_outfile

boolean

Show description in the report

Boolean value Yes/No

-rscoreshow2
-rscoreshow_outfile

boolean

Show the score in the report

Boolean value Yes/No

-rstrandshow2
-rstrandshow_outfile

boolean

Show the nucleotide strand in the report

Boolean value Yes/No

-rusashow2
-rusashow_outfile

boolean

Show the full USA in the report

Boolean value Yes/No

-rmaxall2
-rmaxall_outfile

integer

Maximum total hits to report

Any integer value

-rmaxseq2
-rmaxseq_outfile

integer

Maximum hits to report for one sequence

Any integer value

General qualifiers

-auto

boolean

Turn off prompts

Boolean value Yes/No

-stdout

boolean

Write first file to standard output

Boolean value Yes/No

-filter

boolean

Read first file from standard input, write first file to standard output

Boolean value Yes/No

-options

boolean

Prompt for standard and additional values

Boolean value Yes/No

-debug

boolean

Write debug output to program.dbg

Boolean value Yes/No

-verbose

boolean

Report some/full command line options

Boolean value Yes/No

-help

boolean

Report command line options and exit. More information on associated and general qualifiers can be found with -help -verbose

Boolean value Yes/No

-warning

boolean

Report warnings

Boolean value Yes/No

-error

boolean

Report errors

Boolean value Yes/No

-fatal

boolean

Report fatal errors

Boolean value Yes/No

-die

boolean

Report dying program messages

Boolean value Yes/No

-version

boolean

Report version number and exit

Boolean value Yes/No

Input file format

Any protein sequence.

Input files for usage example

'tsw:opsd_human' is a sequence entry in the example protein database 'tsw'

Database entry: tsw:opsd_human

ID   OPSD_HUMAN              Reviewed;         348 AA.
AC   P08100; Q16414; Q2M249;
DT   01-AUG-1988, integrated into UniProtKB/Swiss-Prot.
DT   01-AUG-1988, sequence version 1.
DT   15-JUN-2010, entry version 128.
DE   RecName: Full=Rhodopsin;
DE   AltName: Full=Opsin-2;
GN   Name=RHO; Synonyms=OPN2;
OS   Homo sapiens (Human).
OC   Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
OC   Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini;
OC   Catarrhini; Hominidae; Homo.
OX   NCBI_TaxID=9606;
RN   [1]
RP   NUCLEOTIDE SEQUENCE [GENOMIC DNA].
RX   MEDLINE=84272729; PubMed=6589631; DOI=10.1073/pnas.81.15.4851;
RA   Nathans J., Hogness D.S.;
RT   "Isolation and nucleotide sequence of the gene encoding human
RT   rhodopsin.";
RL   Proc. Natl. Acad. Sci. U.S.A. 81:4851-4855(1984).
RN   [2]
RP   NUCLEOTIDE SEQUENCE [GENOMIC DNA].
RA   Suwa M., Sato T., Okouchi I., Arita M., Futami K., Matsumoto S.,
RA   Tsutsumi S., Aburatani H., Asai K., Akiyama Y.;
RT   "Genome-wide discovery and analysis of human seven transmembrane helix
RT   receptor genes.";
RL   Submitted (JUL-2001) to the EMBL/GenBank/DDBJ databases.
RN   [3]
RP   NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA].
RC   TISSUE=Retina;
RX   PubMed=17974005; DOI=10.1186/1471-2164-8-399;
RA   Bechtel S., Rosenfelder H., Duda A., Schmidt C.P., Ernst U.,
RA   Wellenreuther R., Mehrle A., Schuster C., Bahr A., Bloecker H.,
RA   Heubner D., Hoerlein A., Michel G., Wedler H., Koehrer K.,
RA   Ottenwaelder B., Poustka A., Wiemann S., Schupp I.;
RT   "The full-ORF clone resource of the German cDNA consortium.";
RL   BMC Genomics 8:399-399(2007).
RN   [4]
RP   NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA].
RX   PubMed=15489334; DOI=10.1101/gr.2596504;
RG   The MGC Project Team;
RT   "The status, quality, and expansion of the NIH full-length cDNA
RT   project: the Mammalian Gene Collection (MGC).";
RL   Genome Res. 14:2121-2127(2004).
RN   [5]
RP   NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-120.
RX   PubMed=8566799; DOI=10.1016/0378-1119(95)00688-5;
RA   Bennett J., Beller B., Sun D., Kariko K.;
RT   "Sequence analysis of the 5.34-kb 5' flanking region of the human
RT   rhodopsin-encoding gene.";


  [Part of this file has been deleted for brevity]

FT                                /FTId=VAR_004816.
FT   VARIANT     209    209       V -> M (effect not known).
FT                                /FTId=VAR_004817.
FT   VARIANT     211    211       H -> P (in RP4; dbSNP:rs28933993).
FT                                /FTId=VAR_004818.
FT   VARIANT     211    211       H -> R (in RP4).
FT                                /FTId=VAR_004819.
FT   VARIANT     216    216       M -> K (in RP4).
FT                                /FTId=VAR_004820.
FT   VARIANT     220    220       F -> C (in RP4).
FT                                /FTId=VAR_004821.
FT   VARIANT     222    222       C -> R (in RP4).
FT                                /FTId=VAR_004822.
FT   VARIANT     255    255       Missing (in RP4).
FT                                /FTId=VAR_004823.
FT   VARIANT     264    264       Missing (in RP4).
FT                                /FTId=VAR_004824.
FT   VARIANT     267    267       P -> L (in RP4).
FT                                /FTId=VAR_004825.
FT   VARIANT     267    267       P -> R (in RP4).
FT                                /FTId=VAR_004826.
FT   VARIANT     292    292       A -> E (in CSNBAD1).
FT                                /FTId=VAR_004827.
FT   VARIANT     296    296       K -> E (in RP4; dbSNP:rs29001653).
FT                                /FTId=VAR_004828.
FT   VARIANT     297    297       S -> R (in RP4).
FT                                /FTId=VAR_004829.
FT   VARIANT     342    342       T -> M (in RP4).
FT                                /FTId=VAR_004830.
FT   VARIANT     345    345       V -> L (in RP4).
FT                                /FTId=VAR_004831.
FT   VARIANT     345    345       V -> M (in RP4).
FT                                /FTId=VAR_004832.
FT   VARIANT     347    347       P -> A (in RP4).
FT                                /FTId=VAR_004833.
FT   VARIANT     347    347       P -> L (in RP4; common variant).
FT                                /FTId=VAR_004834.
FT   VARIANT     347    347       P -> Q (in RP4).
FT                                /FTId=VAR_004835.
FT   VARIANT     347    347       P -> R (in RP4; dbSNP:rs29001566).
FT                                /FTId=VAR_004836.
FT   VARIANT     347    347       P -> S (in RP4; dbSNP:rs29001637).
FT                                /FTId=VAR_004837.
SQ   SEQUENCE   348 AA;  38893 MW;  6F4F6FCBA34265B2 CRC64;
     MNGTEGPNFY VPFSNATGVV RSPFEYPQYY LAEPWQFSML AAYMFLLIVL GFPINFLTLY
     VTVQHKKLRT PLNYILLNLA VADLFMVLGG FTSTLYTSLH GYFVFGPTGC NLEGFFATLG
     GEIALWSLVV LAIERYVVVC KPMSNFRFGE NHAIMGVAFT WVMALACAAP PLAGWSRYIP
     EGLQCSCGID YYTLKPEVNN ESFVIYMFVV HFTIPMIIIF FCYGQLVFTV KEAAAQQQES
     ATTQKAEKEV TRMVIIMVIA FLICWVPYAS VAFYIFTHQG SNFGPIFMTI PAFFAKSAAI
     YNPVIYIMMN KQFRNCMLTT ICCGKNPLGD DEASATVSKT ETSQVAPA
//

Output file format

The output is a standard EMBOSS report file.

The results can be output in one of several styles by using the command-line qualifier -rformat xxx, where 'xxx' is replaced by the name of the required format. The available format names are: embl, genbank, gff, pir, swiss, dasgff, debug, listfile, dbmotif, diffseq, draw, restrict, excel, feattable, motif, nametable, regions, seqtable, simple, srs, table, tagseq.

See: http://emboss.sf.net/docs/themes/ReportFormats.html for further information on report formats.

By default pepdigest writes a 'seqtable' report file.

Output files for usage example

File: opsd_human.pepdigest

########################################
# Program: pepdigest
# Rundate: Fri 15 Jul 2011 12:00:00
# Commandline: pepdigest
#    -seqall tsw:opsd_human
# Report_format: seqtable
# Report_file: opsd_human.pepdigest
########################################

#=======================================
#
# Sequence: OPSD_HUMAN     from: 1   to: 348
# HitCount: 14
#
# Complete digestion with Trypsin yields 14 fragments
#
#=======================================

  Start     End Mol_Weight Cterm  Nterm  Sequence
     70     135   7129.344 R      Y      TPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIER
    178     231   6335.517 R      E      YIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTVK
     22      69   5788.894 R      T      SPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLR
    253     296   5004.103 R      S      MVIIMVIAFLICWVPYASVAFYIFTHQGSNFGPIFMTIPAFFAK
    136     177   4600.476 R      Y      YVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWSR
      1      21   2257.506 .      S      MNGTEGPNFYVPFSNATGVVR
    297     311   1728.097 K      Q      SAAIYNPVIYIMMNK
    232     245   1490.547 K      A      EAAAQQQESATTQK
    326     339   1403.466 K      T      NPLGDDEASATVSK
    315     325   1186.483 R      N      NCMLTTICCGK
    340     348    902.957 K      .      TETSQVAPA
    249     252    503.556 K      M      EVTR
    312     314    449.510 K      N      QFR
    246     248    346.384 K      E      AEK

#---------------------------------------
#---------------------------------------

The header information contains the program name, date of run, name of the reagent used to digest the protein and the number of fragments reported. The header will report if complete or partial digestion was chosen.

The rest of the file consists of columns holding the following data:

The start position of the fragment
The end position of the fragment
The molecular weight of the fragment
The residue before the cut site ('.' if start of sequence)
The residue after the second cut site ('.' if end of sequence)
The sequence of the fragment.

Data files

None.

Notes

If you wish to emulate a partial digestion, then using the -overlap qualifier will display the results from a digest in which all cut sites are used and in which one site at a time is not cut. Thus the resulting peptide fragments from the cut sites numbered 1, 2, 3, 4 etc. are shown, plus the fragments produced by cutting at the sites 1 to 3, 2 to 4, etc. In other words, this option will reveal fragments containing one potential cut site.

If you wish to emulate a very partial digestion (!) then using the -allpartials qualifier will do what -overlap did, but also show all possible fragments from using all possible combinations of cut sites. For example, fragments from the cut sites numbered 1, 2, 3, 4, 5 and from the fragments produced by cutting between sites 1..3, 1..4, 1..5, 2..4, 2..5, 3..5, etc. In other words, this option will reveal fragments containing one or more potential cut site.

If the boolean -ragging is specified then terminal digest fragments are also shown; this can be useful for mass spectrometry. Ragging is further controlled by the -termini option which allows you to select removal of residues from the N and/or C termini.

Each proteolytic enzyme has favoured as well as unfavoured cut sites. For example, Trypsin will not normally cut after a K if it is followed by (e.g.) another K or a P. By default, only data for favoured cut sites is generated. Specifying the qualifier -unfavoured produces data for unfavoured cut sites as well.

References

None.

Warnings

None.

Diagnostic Error Messages

None.

Exit status

It always exits with a status of 0.

Known bugs

None.

Author(s)

Alan Bleasby
European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK

History

Written (1999) - Alan Bleasby

Renamed (2011) from digest to pepdigest to avoid a name clash with another utility.

Target users

This program is intended to be used by everyone and everything, from naive users to embedded scripts.

Comments

None

CSC

Tehdyt toimenpiteet

Wiki

Function

Description

Algorithm

Usage

Command line arguments

Input file format

Input files for usage example

Database entry: tsw:opsd_human

Output file format

Output files for usage example

File: opsd_human.pepdigest

Data files

Notes

References

Warnings

Diagnostic Error Messages

Exit status

Known bugs

See also

Author(s)

History

Target users

Comments