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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.
By default digest uses average molecular weights. The -mono option will use monoisotopic weights.
% digest 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 : Use monoisotopic weights [N]: Output report [opsd_human.digest]:
Go to the input files for this example
Go to the output files for this example
Standard (Mandatory) qualifiers: [-seqall] seqall Protein sequence(s) filename and optional format, or reference (input USA) -menu menu  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 [*.digest] Output report file name 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  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. 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
|Standard (Mandatory) qualifiers||Allowed values||Default|
|Protein sequence(s) filename and optional format, or reference (input USA)||Readable sequence(s)||Required|
|-mono||Use monoisotopic weights||Boolean value Yes/No||No|
|Output report file name||Report output file||<*>.digest|
|Additional (Optional) qualifiers||Allowed values||Default|
|Advanced (Unprompted) qualifiers||Allowed values||Default|
|-mwdata||Molecular weight data for amino acids||Data file||Emolwt.dat|
|-unfavoured||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||No|
|-ragging||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||No|
|-overlap||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||No|
|-allpartials||As for overlap but fragments containing more than one potential cut site are included.||Boolean value Yes/No||No|
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 16-JUN-2009, entry version 116. 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  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  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  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., Blocker H., RA Heubner D., Hoerlein A., Michel G., Wedler H., Kohrer K., RA Ottenwalder 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  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  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). 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). 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). FT /FTId=VAR_004836. FT VARIANT 347 347 P -> S (in RP4). 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 //
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, trace, listfile, dbmotif, diffseq, excel, feattable, motif, regions, seqtable, simple, srs, table, tagseq
See: http://emboss.sf.net/docs/themes/ReportFormats.html for further information on report formats.
By default digest writes a 'seqtable' report file.
######################################## # Program: digest # Rundate: Tue 15 Jul 2008 12:00:00 # Commandline: digest # -seqall tsw:opsd_human # Report_format: seqtable # Report_file: opsd_human.digest ######################################## #======================================= # # 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:
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.
|antigenic||Finds antigenic sites in proteins|
|epestfind||Finds PEST motifs as potential proteolytic cleavage sites|
|fuzzpro||Search for patterns in protein sequences|
|fuzztran||Search for patterns in protein sequences (translated)|
|helixturnhelix||Identify nucleic acid-binding motifs in protein sequences|
|oddcomp||Identify proteins with specified sequence word composition|
|patmatdb||Searches protein sequences with a sequence motif|
|patmatmotifs||Scan a protein sequence with motifs from the PROSITE database|
|pepcoil||Predicts coiled coil regions in protein sequences|
|preg||Regular expression search of protein sequence(s)|
|pscan||Scans protein sequence(s) with fingerprints from the PRINTS database|
|sigcleave||Reports on signal cleavage sites in a protein sequence|