There is an upper limit of 10,000 peaks per individual MS/MS spectrum. Mascot will iteratively select sub-sets of the most intense peaks, looking for the group which most clearly discriminates the score of the top matched protein. In the fragment ion list, the first value is fragment m/z, the second intensity, and the third fragment charge.įragment ion intensity information is very important. They cannot appear within or following the fragment ion list.
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Parameters within an MS/MS query must always be at the beginning, immediately following the BEGIN IONS tag. When an unambiguous charge state can be determined, the correct charge is written to the local CHARGE parameter. For example, the global setting could be 2+ and 3+. This can be useful if the mass spectrometer data system cannot always determine precursor charge state correctly. In the case of the CHARGE parameter, this means that you can have a global CHARGE setting, either from the search form or from a parameter at the head of the data file, as well as a local setting in one or more of the MS/MS queries. Parameters within an MS/MS query only apply locally, to the one spectrum. Precursor intensity and charge can be specified by including additional values on the PEPMASS line, delimited by white space.Ĭertain additional parameters can be specified at query level, between BEGIN IONS and END IONS, as shown in the table below. Within each MS/MS query, the mass of the precursor peptide(s) must be specified using one or more PEPMASS parameters. Parameters specified in the search form or the data file header apply to the entire search. The search form defaults can be over-ridden by including embedded parameters at the beginning of the data file. MS/MS Ions Searchįor an MS/MS Ions Search, each query represents a complete MS/MS spectrum, and is delimited by a pair of statements: BEGIN IONS and END IONS. Sequence queries or MS/MS datasets are not permitted. All of the other search parameters default to the search form settings.Ī peptide mass fingerprint data file can only contain peptide mass fingerprint queries.
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The embedded parameters (COM, CLE, CHARGE, PFA) over-ride the entries in the corresponding form fields, if any. The other is to place embedded parameters at the beginning of the data file. There are two ways to change default search parameters. If your MS data system outputs additional values on each line, these will be ignored. In the case of a Peptide Mass Fingerprint, each query is just a single peptide m/z value, with an optional second value for peak area or intensity. The Mascot generic format for a data file submitted to Mascot is (square brackets indicate optional elements, they should not be included in an actual data file):īlank lines can be used anywhere, to improve readability.Ĭomment lines beginning with one of the symbols # !/ can be included, but only outside of the BEGIN IONS and END IONS statements that delimit an MS/MS dataset.
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The rules which Mascot follows when parsing a data file provide an alternative description of what is and is not acceptable. The following paragraphs illustrate the data file formats by means of examples. The search form fields are essentially defaults for values missing from the data file.
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If there is a conflict between the values of the embedded parameters and values entered into search form fields, the embedded parameters always take precedence. In a Mascot generic format file, certain parameters can appear within an MS/MS dataset. Most embedded parameters can only appear once, at the head of the data file. The following formats are also supported for MS/MS data:Ī data file may include embedded search parameters. In the Mascot generic format, (MGF), each MS/MS dataset is a list of pairs of mass and intensity values, delimited by BEGIN IONS and END IONS statements. In addition, Mascot will automatically recognise the following formats:įor an MS/MS Ions Search, the data file must contain one or more MS/MS peak lists. The peak list formats of a wide range of instrument data systems are directly compatible with these requirements. A Mascot data file is a plain text (ASCII) file containing peak list information and, optionally, search parameters.įor a Peptide Mass Fingerprint, the file should contain a list of peptide mass values, one per line, optionally followed by white space and a peak area or intensity value.