MGE-PortalWiki/EMOWSE

Information about MOWSE

Mowseparameters

You must supply various parameters in order to run Mowse successfully. We have provided default values for some of these parameters; information to help you can be found here.

Databases

We offer several databases for your mowsesearch:

1. Domo

2. Prodom

3. Trembl

4. Sprot

5. Tremblnew

Enzymes

Use this to specify the reagent used for your protein digestion. Mowse currently supports the following reagents:

Reagents	Cleavage rule
Trypsin	C-term to K/R
Lys-C	C-term to K
Arg-C	C-term to R
Asp-N	N-term to D
V8-bicarb	C-term to E
V8-phosph	C-term to E/D
Chymotrypsin	C-term to F/W/Y/L/M
C N Br	C-term to M

Note that cleavage is not allowed if the following residue is proline and that Mowse automatically calculates for nearest-neighbour partial cleavages.

Error 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.

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'. 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.

Filter

If a total molecular weight was given, Mowse will restrict its search to a proteins with that total molecular weight +/- this percentage. 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.

Partial Factors

This specifies the weighting given to partially-cleaved peptide fragments, with a range from 0.1 to 1.0. The factor effectively down-weights the score awarded to a partial fragment by the specified amount. For example, a Pfactor 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 Pfactor (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 Pfactor to optimize searches where the peptide Mw data contain a significant proportion of partial cleavage fragments (eg > 30%). In such cases, setting the Pfactor 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 Pfactor of 0.1 will give maximum discrimination.

Input

The input file is a list of molecular weights of the peptide fragments. One weight is allowed per line.

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.