emowse

 

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Function

Search protein sequences by digest fragment molecular weight

Description

Given an input file of molecular weights corresponding to peptides cut by proteolytic enzymes or reagents, emowse will search the supplied input protein sequences for digest fragments that match the molecular weights. For each input sequence, emowse derives both whole sequence molecular weight and calculated peptide molecular weights for complete digests. One of eight cutting enzymes/reagents can be specified and an optional whole sequence molecular weight (if known). Optionally, monoisotopic weights are used. emowse also incorporate calculated peptide Mw's resulting from incomplete or partial cleavages. At present, this is achieved by computing all nearest-neighbour pairs for each enzyme or reagent.

emowse writes an output file that includes: i. The specified search parameters (digest reagent, specified error tolerance, specified intact protein Mw and Mw filter percentage). ii. Short 'hit' listing (the top 50 scoring proteins listed in descending order, the sequence ID name and brief text identifiers are included). iii. Detailed 'hit' listing (the top 50 entries listed in more detail).

Usage

Here is a sample session with emowse


% emowse 
Search protein sequences by digest fragment molecular weight
Input protein sequence(s): tsw:*
Peptide molecular weight values file: test.mowse
Whole sequence molwt [0]: 
Use monoisotopic weights [N]: 
Output file [cru4_arath.emowse]: 

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

Command line arguments

Search protein sequences by digest fragment molecular weight
Version: EMBOSS:6.5.0.0

   Standard (Mandatory) qualifiers:
  [-sequence]          seqall     Protein sequence(s) filename and optional
                                  format, or reference (input USA)
  [-infile]            infile     Peptide molecular weight values file
   -weight             integer    [0] Whole sequence molwt (Any integer value)
   -mono               boolean    [N] Use monoisotopic weights
  [-outfile]           outfile    [*.emowse] Output file name

   Additional (Optional) qualifiers: (none)
   Advanced (Unprompted) qualifiers:
   -mwdata             datafile   [Emolwt.dat] Molecular weights data file
   -frequencies        datafile   [Efreqs.dat] Amino acid frequencies data
                                  file
   -enzyme             menu       [1] Enzyme or reagent (Values: 1 (Trypsin);
                                  2 (Lys-C); 3 (Arg-C); 4 (Asp-N); 5
                                  (V8-bicarb); 6 (V8-phosph); 7
                                  (Chymotrypsin); 8 (CNBr))
   -pcrange            integer    [25] Allowed whole sequence weight
                                  variability (Integer from 0 to 75)
   -tolerance          float      [0.1] Tolerance (Number from 0.100 to 1.000)
   -partials           float      [0.4] Partials factor (Number from 0.100 to
                                  1.000)

   Associated qualifiers:

   "-sequence" 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
   -scircular1         boolean    Sequence is circular
   -sformat1           string     Input sequence format
   -iquery1            string     Input query fields or ID list
   -ioffset1           integer    Input start position offset
   -sdbname1           string     Database name
   -sid1               string     Entryname
   -ufo1               string     UFO features
   -fformat1           string     Features format
   -fopenfile1         string     Features file name

   "-outfile" associated qualifiers
   -odirectory3        string     Output directory

   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
[-sequence]
(Parameter 1)
seqall Protein sequence(s) filename and optional format, or reference (input USA) Readable sequence(s) Required
[-infile]
(Parameter 2)
infile Peptide molecular weight values file Input file Required
-weight integer Whole sequence molwt Any integer value 0
-mono boolean Use monoisotopic weights Boolean value Yes/No No
[-outfile]
(Parameter 3)
outfile Output file name Output file <*>.emowse
Additional (Optional) qualifiers
(none)
Advanced (Unprompted) qualifiers
-mwdata datafile Molecular weights data file Data file Emolwt.dat
-frequencies datafile Amino acid frequencies data file Data file Efreqs.dat
-enzyme list Enzyme or reagent
1 (Trypsin)
2 (Lys-C)
3 (Arg-C)
4 (Asp-N)
5 (V8-bicarb)
6 (V8-phosph)
7 (Chymotrypsin)
8 (CNBr)
1
-pcrange integer Allowed whole sequence weight variability Integer from 0 to 75 25
-tolerance float Tolerance Number from 0.100 to 1.000 0.1
-partials float Partials factor Number from 0.100 to 1.000 0.4
Associated qualifiers
"-sequence" associated seqall qualifiers
-sbegin1
-sbegin_sequence
integer Start of each sequence to be used Any integer value 0
-send1
-send_sequence
integer End of each sequence to be used Any integer value 0
-sreverse1
-sreverse_sequence
boolean Reverse (if DNA) Boolean value Yes/No N
-sask1
-sask_sequence
boolean Ask for begin/end/reverse Boolean value Yes/No N
-snucleotide1
-snucleotide_sequence
boolean Sequence is nucleotide Boolean value Yes/No N
-sprotein1
-sprotein_sequence
boolean Sequence is protein Boolean value Yes/No N
-slower1
-slower_sequence
boolean Make lower case Boolean value Yes/No N
-supper1
-supper_sequence
boolean Make upper case Boolean value Yes/No N
-scircular1
-scircular_sequence
boolean Sequence is circular Boolean value Yes/No N
-sformat1
-sformat_sequence
string Input sequence format Any string  
-iquery1
-iquery_sequence
string Input query fields or ID list Any string  
-ioffset1
-ioffset_sequence
integer Input start position offset Any integer value 0
-sdbname1
-sdbname_sequence
string Database name Any string  
-sid1
-sid_sequence
string Entryname Any string  
-ufo1
-ufo_sequence
string UFO features Any string  
-fformat1
-fformat_sequence
string Features format Any string  
-fopenfile1
-fopenfile_sequence
string Features file name Any string  
"-outfile" associated outfile qualifiers
-odirectory3
-odirectory_outfile
string Output directory Any string  
General qualifiers
-auto boolean Turn off prompts Boolean value Yes/No N
-stdout boolean Write first file to standard output Boolean value Yes/No N
-filter boolean Read first file from standard input, write first file to standard output Boolean value Yes/No N
-options boolean Prompt for standard and additional values Boolean value Yes/No N
-debug boolean Write debug output to program.dbg Boolean value Yes/No N
-verbose boolean Report some/full command line options Boolean value Yes/No Y
-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 N
-warning boolean Report warnings Boolean value Yes/No Y
-error boolean Report errors Boolean value Yes/No Y
-fatal boolean Report fatal errors Boolean value Yes/No Y
-die boolean Report dying program messages Boolean value Yes/No Y
-version boolean Report version number and exit Boolean value Yes/No N

Input file format

Input files for usage example

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

File: test.mowse

6082.8
5423.0
3086.3
2930.3
2424.7
2030.2
1399.6
1086.2

The input file is a list of molecular weights of the peptide fragments. One weight is allowed per line. The example file above is a Trypsin digest of the protein sw:100K_rat (produced by using the program digest).

Each molecular weight must be on a line of its own. Masses (M not M[H+]) are accepted in any order (ascending,descending or mixed). Peptide masses can be entered as integers or floating-point values, the latter being rounded to the nearest integer value for the search.

It is suggested that peptide masses should be selected from the range 700-4000 Daltons. This range balances the fact that very small peptides give little discrimination and minimizes the frequency of partially-cleaved peptides.

As a general rule, users are advised to identify and remove peptide masses resulting from autodigestion of the cleavage enzyme (e.g tryptic fragments of trypsin), best obtained by MS analysis of control digests containing only the enzyme.

Further information on the partial sequence and/or composition of the peptides can be given after the weight with a 'seq()' or 'comp()' specification. This should be formatted like:

mw seq(...) comp(...)

where mw is the molecular mass of the fragment, seq(...) is sequence information and comp(...) is composition information. A line may contain more than one sequence information qualifiers. For example:


7176 seq(b-t[pqt]ln)
1744
1490
1433   comp(3[ed]1[p]) seq(gmde)

Sequence information

The sequence information should be given in standard One-character code. It should be preceded by a prefix as outlined in the table below, to indicate what type of sequence it is.

Prefixes to use with sequence information for emowse
PrefixMeaningExample
b-N->C sequence seq(b-DEFG)
y-C->N sequence seq(y-GFED)
*- Orientation unknown seq(*-DEFG)
seq(*-GFED)
n-N terminal sequence seq(n-ACDE)
c-C terminal sequence seq(c-FGHI)
The examples are all correct data for a peptide with a sequence ACDEFGHI.
Note that *-DEFG will search for both DEFG and GFED

Both lower and upper case characters may be used for amino-acids. An unknown amino acid may be indicated by an 'X'. More than one amino acid may be specified for a position by putting them between square brackets. A line may contain several sequence information qualifiers. An example for a peptide with the actual sequence ACDEFGHI might look like:

12345 seq(n-AC[DE]) seq(c-HI)

Composition Information

Composition should consist of a number, followed by the corresponding amino acid between square brackets. For example
comp(2[H]0[M]3[DE]*[K])
indicates a peptide which contains 2 histidines, no methionines, 3 acidic residues (glutamic or aspartic acid) and at least 1 lysine.

Output file format

Output files for usage example

File: cru4_arath.emowse


Using data fragments of:
          1086.2  
          1399.6  
          2030.2  
          2424.7  
          2930.3  
          3086.3  
          5423.0  
          6082.8  

1   UBR5_RAT     E3 ubiquitin-protein ligase UBR5 (6.3.2.-) (100 kDa protein) (
2   SYVC_TAKRU   Valine--tRNA ligase (6.1.1.9) (Valyl-tRNA synthetase) (ValRS) 
3   TCPD_TAKRU   T-complex protein 1 subunit delta (TCP-1-delta) (CCT-delta)   
4   OPS2_DROME   Opsin Rh2 (Ocellar opsin)                                     
5   FLAV_ECO57   Flavodoxin-1                                                  
6   FLAV_ECOL6   Flavodoxin-1                                                  
7   FLAV_ECOLI   Flavodoxin-1                                                  
8   FLAV_KLEPN   Flavodoxin                                                    
9   FLAV_SYNY3   Flavodoxin                                                    
10  EI2BB_TAKRU  Translation initiation factor eIF-2B subunit beta (S20I15) (eI
11  FLAV_HAEIN   Flavodoxin                                                    
12  HIRA_TAKRU   Protein HIRA (TUP1-like enhancer of split protein 1)          
13  OPS2_SCHGR   Opsin-2                                                       
14  LACY_ECOLI   Lactose permease (Lactose-proton symport)                     
15  FLAV_CLOSA   Flavodoxin                                                    
16  AMIC_PSEAE   Aliphatic amidase expression-regulating protein               
17  PAX3_HUMAN   Paired box protein Pax-3 (HuP2)                               
18  PAX4_HUMAN   Paired box protein Pax-4                                      
19  CO9_TAKRU    Complement component C9 (Precursor)                           
20  SYHC_TAKRU   Histidine--tRNA ligase, cytoplasmic (6.1.1.21) (Histidyl-tRNA 
21  BGAL_ECOLI   Beta-galactosidase (Beta-gal) (3.2.1.23) (Lactase)            
22  HD_TAKRU     Huntingtin (Huntington disease protein homolog) (HD protein ho

    1  : UBR5_RAT       1.212e+05 308026.9   0.750 
         E3 ubiquitin-protein ligase UBR5 (6.3.2.-) (100 kDa protein) (E3 ubiquitin-protein ligase, HECT domain-containing 1) (Hyperplastic discs protein homolog)
         Mw     Start  End    Seq
         1086.3 2257   2266   CATTPMAVHR                                   
         1399.6 1905   1916   GDFLNYALSLMR                                 
         2424.7 2189   2211   VFMEDVGAEPGSILTELGGFEVK                      
         2930.3 2570   2597   QLILASQSSDADAVFSAMDLAFAVDLCK                 
         3086.3 2357   2384   QLSIDTRPFRPASEGNPSDDPDPLPAHR                 
        *6082.9 2716   2769   QDLVYFWTSSPSLPASEEGFQPMPSITIRPPDDQHLPTANTCISR...
         No Match      2030.2 5423.0 

    2  : SYVC_TAKRU     3.791e+01 138218.2   0.375 
         Valine--tRNA ligase (6.1.1.9) (Valyl-tRNA synthetase) (ValRS)
         Mw     Start  End    Seq
         1087.3 510    518    TVLHPFCDR                                    
        *1399.6 1184   1195   VPVKVQEQDTEK                                 


  [Part of this file has been deleted for brevity]

         No Match      1086.2 1399.6 2030.2 2424.7 2930.3 5423.0 6082.8 

    15 : FLAV_CLOSA     4.938e+00 17763.4    0.125 
         Flavodoxin
         Mw     Start  End    Seq
        *1085.3 17     26     VAKLIEEGVK                                   
         No Match      1399.6 2030.2 2424.7 2930.3 3086.3 5423.0 6082.8 

    16 : AMIC_PSEAE     3.859e+00 42807.1    0.125 
         Aliphatic amidase expression-regulating protein
         Mw     Start  End    Seq
        *2423.7 308    328    VEDVQRHLYDICIDAPQGPVR                        
         No Match      1086.2 1399.6 2030.2 2930.3 3086.3 5423.0 6082.8 

    17 : PAX3_HUMAN     3.494e+00 52967.5    0.125 
         Paired box protein Pax-3 (HuP2)
         Mw     Start  End    Seq
        *2930.4 11     37     MMRPGPGQNYPRSGFPLEVSTPLGQGR                  
         No Match      1086.2 1399.6 2030.2 2424.7 3086.3 5423.0 6082.8 

    18 : PAX4_HUMAN     3.488e+00 37832.7    0.125 
         Paired box protein Pax-4
         Mw     Start  End    Seq
        *2029.4 28     45     QQIVRLAVSGMRPCDISR                           
         No Match      1086.2 1399.6 2424.7 2930.3 3086.3 5423.0 6082.8 

    19 : CO9_TAKRU      3.007e+00 65197.9    0.125 
         Complement component C9 (Precursor)
         Mw     Start  End    Seq
        *2930.2 135    162    TCPPTVLDTNEQGRTAGYGINILGADPR                 
         No Match      1086.2 1399.6 2030.2 2424.7 3086.3 5423.0 6082.8 

    20 : SYHC_TAKRU     2.821e+00 57913.0    0.125 
         Histidine--tRNA ligase, cytoplasmic (6.1.1.21) (Histidyl-tRNA synthetase) (HisRS)
         Mw     Start  End    Seq
         1087.2 124    133    DQGGELLSLR                                   
         No Match      1399.6 2030.2 2424.7 2930.3 3086.3 5423.0 6082.8 

    21 : BGAL_ECOLI     2.280e+00 116482.9   0.125 
         Beta-galactosidase (Beta-gal) (3.2.1.23) (Lactase)
         Mw     Start  End    Seq
         1400.6 601    612    QFCMNGLVFADR                                 
         No Match      1086.2 2030.2 2424.7 2930.3 3086.3 5423.0 6082.8 

    22 : HD_TAKRU       2.169e+00 348936.6   0.375 
         Huntingtin (Huntington disease protein homolog) (HD protein homolog)
         Mw     Start  End    Seq
        *1400.6 2899   2911   VDGEALVKLSVDR                                
        *2031.3 645    663    LLSASFLLTGQKNGLTPDR                          
        *3085.6 1573   1597   LVQYHQVLEMFILVLQQCHKENEDK                    
         No Match      1086.2 2424.7 2930.3 5423.0 6082.8 

The emowse search program outputs a listing file containing the following information.

Specified search parameters

Includes all specified parameters such as digest reagent, specified error tolerance, specified intact protein Mw and Mw filter percentage. All supplied peptide Mws are listed in descending order, followed by the total number of entries scanned during the search.

Short 'hit' listing

The top 50 scoring proteins are then listed in descending order, details include the sequence ID name and brief text identifiers. Details are limited to the top 50 scores as a deliberate compromise to keep the result listings as short as possible.

Detailed 'hit' listing

The top 50 entries are then listed in more detail.The first line includes the sequence ID name, the emowse search score (typically a few powers of 10), the 'hit' protein Mw and finally an 'accuracy' ratio calculated by dividing 'hits' by the total number of peptides used for the search. This cannot be used to ascribe significance, but experience has shown that anything below 0.3 is generally not worth pursuing. Line 2 is the protein text identifier. Subsequent lines list 'hit' and 'miss' peptides, with the appropriate start, end and corresponding sequences of correct peptide matches. 'miss' peptides are indicated by 'No match' at the start of the last line for that protein.

Matching peptides marked with a '*' denote partially-cleaved fragments.

Data files

emowse reads in the pre-computed "Frequencies" data file 'Efreqs.dat', (See the section "emowse Scoring scheme", above for a description of the frequency scores.)

EMBOSS data files are distributed with the application and stored in the standard EMBOSS data directory, which is defined by the EMBOSS environment variable EMBOSS_DATA.

To see the available EMBOSS data files, run:

% embossdata -showall

To fetch one of the data files (for example 'Exxx.dat') into your current directory for you to inspect or modify, run:


% embossdata -fetch -file Exxx.dat

Users can provide their own data files in their own directories. Project specific files can be put in the current directory, or for tidier directory listings in a subdirectory called ".embossdata". Files for all EMBOSS runs can be put in the user's home directory, or again in a subdirectory called ".embossdata".

The directories are searched in the following order:

Notes

Peptide mass information can provide a 'fingerprint' signature sufficiently discriminating to allow for the unique and rapid identification of unknown sample proteins, independent of other analytical methods such as protein sequence analysis. Practical experience has shown that sample proteins can be uniquely identified using as few as 3-4 experimentally determined peptide masses when screened against a fragment database derived from over 50,000 proteins.

Given a one-per-line file of molecular weights cut by enzymes/reagents, emowse will search a protein database for matches with the mass spectrometry data.

One of eight cutting enzymes/reagents can be specified and an optional whole sequence molecular weight.

Determination of molecular weight has always been an important aspect of the characterization of biological molecules. Protein molecular weight data, historically obtained by SDS gel electrophoresis or gel permeation chromatography, has been used establish purity, detect post-translational modification (such as phosphorylation or glycosylation) and aid identification. Until just over a decade ago, mass spectrometric techniques were typically limited to relatively small biomolecules, as proteins and nucleic acids were too large and fragile to withstand the harsh physical processes required to induce ionization. This began to change with the development of 'soft' ionization methods such as fast atom bombardment (FAB)[1], electrospray ionisation (ESI) [2,3] and matrix-assisted laser desorption ionisation (MALDI)[4], which can effect the efficient transition of large macromolecules from solution or solid crystalline state into intact, naked molecular ions in the gas phase. As an added bonus to the protein chemist, sample handling requirements are minimal and the amounts required for MS analysis are in the same range, or less, than existing analytical methods.

As well as providing accurate mass information for intact proteins, such techniques have been routinely used to produce accurate peptide molecular weight 'fingerprint' maps following digestion of known proteins with specific proteases. Such maps have been used to confirm protein sequences (allowing the detection of errors of translation, mutation or insertion), characterise post-translational modifications or processing events and assign disulphide bonds [5,6].

Less well appreciated, however, is the extent to which such peptide mass information can provide a 'fingerprint' signature sufficiently discriminating to allow for the unique and rapid identification of unknown sample proteins, independent of other analytical methods such as protein sequence analysis.

Practical experience has shown that sample proteins can be uniquely identified using as few as 3- 4 experimentally determined peptide masses when screened against a fragment database derived from over 50,000 proteins. Experimental errors of a few Daltons are tolerated by the scoring algorithms, permitting the use of inexpensive time-of-flight mass spectrometers. As with other types of physical data, such as amino acid composition or linear sequence, peptide masses can clearly provide a set of determinants sufficiently unique to identify or match unknown sample proteins. Peptide mass fingerprints can prove as discriminating as linear peptide sequence, but can be obtained in a fraction of the time using less material. In many cases, this allows for a rapid identification of a sample protein before committing to protein sequence analysis. Fragment masses also provide structural information, at the protein level, fully complementary to large-scale DNA sequencing or mapping projects [7,8,9].

For each entry in the specified set of sequences to search, emowse derives both whole sequence molecular weight and calculated peptide molecular weights for complete digests using the range of cleavage reagents and rules detailed in Table 1. Cleavage is disallowed if the target residue is followed by proline (except for CNBr or Asp N). Glu C (S. aureus V8 protease) cleavages are also inhibited if the adjacent residue is glutamic acid. Peptide mass calculations are based entirely on the linear sequence and use the average isotopic masses of amide-bonded amino acid residues (IUPAC 1987 relative atomic masses). To allow for N-terminal hydrogen and C-terminal hydroxyl the final calculated molecular weight of a peptide of N residues is given by the equation:

        N
        __
        \
        /  Residue mass + 18.0153
        --
        n=1        
Molecular weights are rounded to the nearest integer value before being used. Cysteine residues are calculated as the free thiol, anticipating that samples are reduced prior to mass analysis. CNBr fragments are calculated as the homoserine lactone form. Information relating to post- translational modification (phosphorylation, glycosylation etc.) is not incorporated into calculation of peptide masses.

Table 1: Cleavage reagents modelled by emowse.

Reagent no.     Reagent                 Cleavage rule   
                                
        1       Trypsin                 C-term to K/R
        2       Lys-C                   C-term to K
        3       Arg-C                   C-term to R
        4       Asp-N                   N-term to D
        5       V8-bicarb               C-term to E
        6       V8-phosph               C-term to E/D
        7       Chymotrypsin            C-term to F/W/Y/L/M
        8       CNBr                    C-term to M

Current versions of emowse also incorporate calculated peptide Mw's resulting from incomplete or partial cleavages. At present, this is achieved by computing all nearest-neighbour pairs for each enzyme or reagent detailed in table 1.

Tolerance

The supplied number specifies the error allowed for mass accuracy of experimental mass determination. If no figure is specified, a default tolerance of 2 Daltons will be assumed. If you wish to specify a different tolerance then follow the qualifier '-tolerance' with the required number of Daltons. eg: '-tolerance 1'. In this case, supplied peptide masses will be matched to +/- 1 Daltons. Values of 2-4 are suggested for data obtained by laser- desorption TOF instruments. Accuracies of +/- 2 Daltons or better are generally only possible using an appropriate internal standard (e.g. oxidised insulin B chain) with TOF instruments. For electrospray or FAB data, a value of 1 can be selected in most cases. If you have real confidence in mass determination, specify '0' (zero) to limit matches to the nearest integer value (effectively +/- 0.5 Daltons). Discrimination is significantly improved by the selection of a small error tolerance.

Whole sequence molecular weight

This option allows you to give the molwt of the whole protein (if known). This allows you to limit the search to proteins of this molwt plus/minus a 'limit' (see below). If unspecified, a whole protein molwt of 0 is assumed which emowse interprets as "search the whole database". This will include all proteins up to the maximum size of just under 700,000 Daltons. You can specify any molwt in Daltons with this command e.g. '-weight 90000'.

Allowed whole sequence weight variability

This option is used in conjunction with the '-weight' option and is meaningless without it. It specifies a percentage. Only proteins of the given Sequence molecular weight +/- this percentage will be searched. If a Sequence molecular weight is specified but '-pcrange' is unspecified then '-pcrange ' will default to 25%. To specify a percentage of 30% use: '-pcrange 30'. In this case, a molecular weight of 90,000 Daltons was specified and the selection of 30 for the filter restricts the search to those proteins with masses from 63,000 to 117,000 Daltons. A value of 25 is suggested for initial searches, which can be progressively widened for subsequent search attempts if no matches are found. Discrimination is best when the filter percentage is narrow, but some Mw estimates (particularly from SDS gels) should be given considerable allowance for error.

Partials factor

This specifies the weighting given to partially-cleaved peptide fragments, with a range from 0.1 to 1.0. If not specified, the default value is 0.4. The factor effectively down-weights the score awarded to a partial fragment by the specified amount. For example, a '-partials' of 0.25 will reduce the score of partial fragments to 25% (one quarter) of the score of a complete ('perfect') peptide cleavage fragment of equal mass.

Computing all possible nearest-neighbour partial fragments adds significantly to the number of peptides entered in the database (by a factor of two). The major effect of this is to increase the background score by increasing the number of random Mw matches, which can significantly reduce discrimination. The use of a low '-partials' factor (eg 0.1 - 0.3) is a useful way of limiting this effect - partial peptide matches will add a little to the cumulative frequency score, but without compromising discrimination.

More experienced users can utilise the '-partials' factor to optimize searches where the peptide Mw data contain a significant proportion of partial cleavage fragments (eg > 30%). In such cases, setting the '-partials' factor within the range 0.4 - 0.6 can help to improve discrimination. Conversely, if the digestion is perfect, with no partial fragments present, the lowest '-partials' factor of 0.1 will give maximum discrimination.

Program requirements

The emowse search program accepts a single text file containing a list of experimentally-determined masses, generally selected from the range 700-4,000 Daltons to reduce the influence of partial cleavage products. The program outputs a ranked hit list comprising the top 30 scores, with information including the protein entry name, text identifiers, final accumulated scores, matching peptide sequences and hit versus miss tallies. User-selectable search parameters include an error tolerance (default +/- 2 Daltons), selection of the enzyme or reagent used and an intact protein Mw (optional, if known).

For each peptide Mw entry in the data file, emowse matches individual fragment molecular weights (FMWs) with database entry molecular weights (DBMWs). A 'hit' is scored when the following criterion is met:

        DBMW-tolerance-1 < FMW < DBMW+tolerance+1

If an intact protein Mw is specified (SMW) then the program prompts for a molecular weight filter percentage (MWFP). emowse then restricts the search to those entries which match the following criteria:

        R = SMW x MWFP / 100
        0 < SMW-R < emowse entry Mol.wt. < SMW+R

Default search parameters are a tolerance of +/- 2 Daltons, intact Mw specified and the MWFP set to 25.

emowse Scoring scheme

The final scoring scheme is based on the frequency of a fragment molecular weight being found in a protein of a given range of molecular weight. OWL database sequence entries were initially grouped into 10 kDalton intact molecular weight intervals. For each 10 kDalton protein interval, peptide fragment molecular weights were assigned to cells of 100 Dalton intervals. The cells therefore contained the number of times a particular fragment molecular weight occurred in a protein of any given size. This operation was performed for each enzyme. Cell frequency values were calculated by dividing each cell value by the total number of peptides in each 10 kD protein interval. Cell frequency values for each 10 kDalton interval were then normalised to the largest cell value (Fmax), with all the cell values recalculated as:

        Cell value = Old value / Fmax

to yield floating point numbers between 0 and 1. These distribution frequency values, calculated for each cleavage reagent, were then built into the emowse search program. For every database entry scanned, all matching fragments contribute to the final score. In the current implementation, non-matching fragments are ignored (neutral). For each matching peptide Mw a score is assigned by looking up the appropriate normalised distribution frequency value. In the case of multiple 'hits' in any one target protein (i.e. more than one matching peptide Mw), the distribution frequency scores are multiplied. The final product score is inverted and then normalised to an 'average' protein Mw of 50 kDaltons to reduce the influence of random score accumulation in large proteins (>200 kDaltons). The final score is thus calculated as:

Score = 50/(Pn x H)

Where Pn is the product of n distribution scores and H the 'hit' protein molecular weight in kD.

Important consequences of this type of scoring scheme are that matches with peptides of higher Mw carry more scoring weight, and that the non-random distribution of fragment molecular weights in proteins of different sizes is compensated for.

Simulation studies

In a simulation of scoring properties, 100 test proteins with masses between 10 kD and 100 kD were randomly selected from the OWL sequence database. The sets of all possible tryptic peptide masses for each protein were randomized and database searches performed with increasing numbers of fragments (default search parameters) until the test protein reached the top of the ranked scoring list. 99% of the test proteins were correctly identified using only five peptides or less (mean=3.6 peptides), with one example requiring six. These figures were surprisingly small considering that some of the proteins in the test sample generated more than 100 possible tryptic fragments. All 100 test examples were identified using 30% or less of the maximum number of available peptides.

This distribution was somewhat dependent on protein size, as smaller proteins generally yield fewer peptide fragments. Thus, all proteins of 30 kD and over were identified using 13% or less of possible fragments (1 in 8), with all proteins of 40 kD and above requiring less than 10% (1 in 10). In this latter group, therefore, more than 90% of the potential information (all possible peptides) was redundant. For the simulation a 'unique' identification required matching not only of protein type (e.g. globin) but correct discrimination of type, species, and isoform or isozyme. Discrimination could be further improved by reducing the error tolerance to only +/- 1 Dalton (mean=2.7 peptides). Such accuracies are easily bettered using more sophisticated ESI/quadrupole or high-field FAB spectrometers, but the default search value (+/- 2 Daltons) compensates for the reduced accuracy obtainable from the smaller time-of-flight (TOF) instruments. Mass accuracies better than +/- 1 Dalton were not essential, and in fact the error tolerance could be relaxed to +/- 5 Daltons in many cases with little degradation in performance. The simulation thus clearly demonstrated the high degree of discrimination afforded by relatively few peptide masses, even with generous allowance for error.

References

    The paper describing the original 'MOWSE' program is:
  1. D.J.C. Pappin, P. Hojrup and A.J. Bleasby 'Rapid Identification of Proteins by Peptide-Mass Fingerprinting'. Current Biology (1993), vol 3, 327-332.

    Other references:

  2. Barber M, Bordoli RS, Sedgwick RD, Tyler AN: Fast atom bombardment of solids: a new ion source for mass spectrometry. J Chem Soc Chem Commun 1981, 7: 325-327.
  3. Dole M, Mack LL, Hines RL, Mobley RC, Ferguson LD, Alice MB: Molecular beams of macroions. J Chem Phys 1968, 49:2240-2249.
  4. Meng CK, Mann M, Fenn JB: Of protons or proteins. Z Phys D 1988, 10: 361-368.
  5. Karas M, Hillenkamp F: Laser desorption ionisation of proteins with molecular masses exceeding 10,000 Daltons. Analytical Chemistry 1988, 60:2299-2301.
  6. Morris H, Panico M, Taylor GW: FAB-mapping of recombinant-DNA protein products. Biochem Biophys Res Commun 1983, 117:299-305.
  7. Morris H, Greer FM: Mass spectrometry of natural and recombinant proteins and glycoproteins. Trends in Biotechnology 1988, 6:140-147.
  8. Weissenbach J, Gyapay G, Dib C, Vignal J, Morissette J, Millasseau P, Vaysseix G, Lathrop M: A second generation linkage map of the human genome. Nature 1992, 359:794-801.
  9. Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF, Kerlavage AR, McCombie WR, Venter JC: Complementary DNA sequencing: expressed sequence tags and human genome project. Science 1991, 252:1651-1656.
  10. Lehrach H, Drmanac R, Hoheisel J, Larin Z, Lennon G, Monaco AP, Nizetic D, Zehetner G, Poustka A: Hybridization fingerprinting in genome mapping and sequencing. In Genome Analysis Volume 1: Genetic and Physical Mapping. Cold Spring Harbor Laboratory Press; 1990:39-81 .
  11. Akrigg D, Bleasby AJ, Dix NIM, Findlay JBC, North ACT, Parry- Smith D, Wootton JC, Blundell TI, Gardner SP, Hayes F, Sternberg MJE, Thornton JM, Tickle IJ, Murray-Rust P: A protein sequence/structure database. Nature 1988, 335:745-746.
  12. Bleasby AJ, Wootton JC: Construction of validated, non- redundant composite protein databases. Protein Engineering 1990, 3:153-159.

Warnings

None.

Diagnostic Error Messages

None.

Exit status

It always exits with status 0.

Known bugs

None.

See also

Program name Description
backtranambig Back-translate a protein sequence to ambiguous nucleotide sequence
backtranseq Back-translate a protein sequence to a nucleotide sequence
compseq Calculate the composition of unique words in sequences
freak Generate residue/base frequency table or plot
mwcontam Find weights common to multiple molecular weights files
mwfilter Filter noisy data from molecular weights file
oddcomp Identify proteins with specified sequence word composition
pepdigest Report on protein proteolytic enzyme or reagent cleavage sites
pepinfo Plot amino acid properties of a protein sequence in parallel
pepstats Calculate statistics of protein properties
wordcount Count and extract unique words in molecular sequence(s)

Author(s)

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

Please report all bugs to the EMBOSS bug team (emboss-bug © emboss.open-bio.org) not to the original author.

History

Written (Sept 2000) - Alan Bleasby.

Target users

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

Comments

None