3. Importing search results
Importing search results is a great way to quickly view spectra of interest and assess the quality of peptide-spectrum-matches (PSMs).
To import a search results file, click the SQLite icon (make database) in the toolbar. This makes a sqlite database from an excel file containing Mascot, COMET, or X!Tandem search results (in multiplierz format; see below section on “Search Result Compatibility”). All rows will be imported into a sqlite file in a table called “peptides”. All queries should be performed against this table. mzStudio can also import PSM files from Proteome Discoverer, see the section below "Importing Proteome Discoverer Search Results".
-
Note, all search results MUST be in multiplierz format to be read correctly! mzStudio looks for specific worksheets (‘Mascot_Header’ for a Mascot Search and ‘Data’ for PSMs) and specific columns (i.e. ‘Spectrum Description’). An incorrectly formatted file will throw errors or cause unexpected behavior.
-
After loading the results, they are displayed in grid format. An mzPepCalc window is also opened automatically.
- Clicking on a row label jumps to the selected scan and highlights that row. The sequence is displayed in the mzPepCalc sequence box and the fragment ions are calculated. In addition, fragment ions are labeled in the spectrum window.
- Search results may be filtered and sorted using SQLite queries. Valid queries can be entered into the text box to the right of the ‘Submit’ button, which is pressed to apply the query. The text displayed initially in this box (‘select * from peptides’) is the query used to generate the initial data table.
Example SQLite queries:
- To sort data from highest to lowest scoring peptides, enter:
select * from peptides order by "Peptide Score" desc and click submit.
- To display only oxidized methionine containing peptides, enter:
select * from peptides where “Variable Modifications” like “%Oxidation%”
- To display oxidized methionine peptides which Mascot scores > 30 and sort from highest to lowest mz:
select * from peptides where "Variable Modifications" like "%Oxidation%" and "Mascot Score" > 30 order by "Experimental mz" desc
- Note the autocompletion feature of SQLITE keywords (e.g. SELECT, FROM, WHERE, etc.) which helps query construction. In addition, we implemented shortcuts for common worksheet column names; when these keys are typed, they will be replaced in-line with the following column names:
| Shortcut | For Text |
|---|---|
| ~var | Variable modifications |
| ~sc | Peptide score |
| ~gn | Gene name |
| ~desc | Protein Description |
| ~acc | Accession |
| ~lk | “LIKE %%” |
| ~xc | “Cross correlation” |
| ~ex | Expect |
| ~seq | Peptide Sequence |
To correctly load search results, interpret modifications, and navigate scan information, Mascot, COMET, and X!Tandem results should be in multiplierz format. For more information on using multiplierz/mzDesktop, please refer to the wiki pages:
https://github.com/BlaisProteomics/mzDesktop/wiki https://github.com/BlaisProteomics/multiplierz/wiki
mzStudio requires a header worksheet listing fixed modifications (variable mods are usually listed with the PSMs). In addition, mzStudio looks for a column called “Spectrum Description” with a specific format that it can parse the scan number.
mzStudio can also import text files containing Proteome Discoverer PSMs. To export the PSMs from Proteome Discoverer, select File/Export/To Text (tab delimited):
Check PSMs, then click Export.
If a Thermo rawfile was directly extracted and searched with Proteome Discoverer, the exported PSM file can be directly read by mzStudio.
To search ABSciex data files with Proteome Discoverer, native files must first be converted to .mgf using the ABSciex MS data converter or multiplierz. Unfortunately, PSM exports from .mgf files do not contain scan numbers; "First Scan" instead reports the index of the spectrum within the mgf file. To look up the actual scan number, mzStudio requires the .mgf file that was searched (user will be prompted after selecting PSM file).