Emowse - Protein Identification by mass spectrometry

Overview

For the unique and rapid identification of unknown sample proteins, a 'fingerprint' signature derived from peptide mass information is sufficient. By using the molecular weights of the peptides obtained by mass spectrometry, emowse searches a protein database for matches with the given data. The scoring algorithm tolerates experimental errors of a few Daltons. For each entry in the database to search, emowse derives whole sequence molecular weight and calculated peptide molecular weights for complete digests using a range of cleavage rules. If you like to look for your unknown sample using emowse, the peptide molecular weights of the protein are sufficient. In addition you can specify a variety of other parameters such as cleavage enzyme, whole sequence molecular weight and error tolerance.

How to use

If you like to search for your protein with emowse against a database, you have to select your Experiment and choose the link Mass Spectra Data on the left hand side of the web-page. There you have to select MS for scan type and choose your scans. Afterwards you get on to the search form by clicking on the Peptide Mass Fingerprint link. Now you can specify some details for your search. You can title the search if you like. You can choose any taxonomy but it will not affect the search with emowse. The selection of the searchengine emowse and the database(s) to search against are mandatory. If you know the whole sequence molecular weight of your protein you can declare the weight in the field Protein Mass. The unit is dalton. You can also select the enzyme or reagent used for the cleavage of the protein. If you are unsure which parameters you should specify in addition, do not do anything more. Otherwise you can specify the allowed whole seqence weight variability (Weight Variability), the error allowed for mass accuracy of experimental mass determination (Error Tolerance) and the weighting given to partially-cleaved peptide fragments. See more details in the Use of Parameters section. If you like to repeat a search with the same parameters, you have to choose the search again with old search parameters option at the bottom. All you have to do now is to click the Search button and emowse will do the rest. When the search is finished, you will see a new window with the number of hits. To see the results, click on Browse Results.

Use of Parameters

Databases

The offered databases are provided from the mascot server. You can select as many databases as you like. Emowse will search against each databases individually.

Enzyme/Reagent

Emowse provides the search with the following enzymes/reagents:

Trypsin
Lys-C
Arg-C
Asp-N
V8-bicarb
V8-phosph
Chymotrypsin
CNBr

You can only choose one enzyme/reagent for the searches. Emowse simulates the complete digests for the sequences in the databases using the following rules:

Reagent         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
CNBr            C-term to M

Protein Mass

Here you can specifiy the whole sequence molecular weight (in dalton) for the given protein (if known). If no value is given, emowse will assign 0. This options allows you to limit the search to proteins of this molecular weight plus/minus a 'limit' (see Weight Variability). Otherwise the whole database will be searched.

Weight Variability

Here you can specify the allowed whole sequence weight variability. This option is meaningless without the Protein Mass parameter. If a molecular weight for the protein is specified, this option will limit the search to proteins with molecular weights +/- the given percentage. The default value is 25%. This value is suggested for initial searches, because discrimination is best when the filter percentage is narrow, but some molecular weight estimates (particularly from SDS gels) should be given considerable allowance for error. You can also specify a range of percentages for which searches are done. Example:

If you specify 25% to 30% for Weight Variability, emowse will search each sample 6 times with different percentages for the allowed whole sequence molecular weight:
 * 25%
 * 26%
 * 27%
 * 28%
 * 29%
 * 30%

Error Tolerance

Here you can specify the error allowed for mass accuracy of experimental mass determination. If no value is specified, a default tolerance of 2 Daltons will be assumed. Be aware that descrimination will be improved by selecting a small error tolerance. You can also specify a range of error tolerances to search. Emowse will look for every tolerance given in steps of 0.1.

Partials Factor

Here you can specify the weighting given to parially-cleaved peptide fragments. The default value for Partials Factor is 0.4. This factor down-weights the score awarded to a partial fragment by the given amount.Example:

 A partials factor of 0.4 will reduce the score of partial fragments to 40% of the score of a complete peptide cleavage fragment of equal mass.

If the digestion is perfect (no partial fragments), you can optimize your search by specifiing a low partial factor. But if your data contains a significant proportion of partial cleavage fragments, you should set the partials factor to a higher value to improve discrimination. As the parameters Weight Variabilit and Error Tolerance you can specify a range of partials factors as well. In steps of 0.1, emowse will search with every partials factor given in the range.

Author: Anna-Lena Kranz

ProDBWiki/WebDocumentation/PeptideMassFingerprintWithEmowse