AB SCIEX TOF/TOF
The AB SCIEX TOF/TOF instruments are unusual in that the spectra are
stored in an Oracle database, rather than "raw" files.
There are several options available for searching TOF/TOF data with Mascot Server and it is possible export data to
mzML, which enables Mascot Distiller to be used for quantitation.
AB Sciex GPS Explorer can be used to submit searches direct to an in-house Mascot Server.
AB Sciex provide two software packages to support their TOF/TOF
instruments: 4/5000 Series Explorer and GPS Explorer. In addition, individual spectra
can be exported as *.T2D files and opened by Data
Explorer or Mascot
TS2Mascot is a free utility that extracts a peak list from the Oracle database and loads it into
the Mascot search form in a web browser. This is very convenient for an iTRAQ quantitation experiment.
MS Data Converter is the AB Sciex command line utility to export data in mzML and MGF formats.
An MGF file is the most compact format for submission to Mascot. The mzML format is more verbose, but has the
advantage that it can be opened in Mascot Distiller for searching, de novo sequencing, and quantitation
GPS Explorer includes extensive Mascot integration. Search parameters are defined as part of
an Analysis in GPS Sample Setup. On completion of each search, the results are imported
into the 4000 series database and can be viewed and reported using the GPS Results Browser.
The workflow supports both ICAT® and iTRAQTM quantitation.
If you wish to submit a search from a web browser using the Mascot search form, the 4/5000
Series Explorer has a function called Peaks to Mascot that exports a peak list to an MGF
A free utility called TS2Mascot provides a convenient way to submit a search through the standard Mascot search form.
This is a Windows program that communicates with the Oracle
database using ODBC. The PC will need to have Oracle client software installed,
including the Oracle ODBC driver. To submit a Mascot search from TS2Mascot, Microsoft
Internet Explorer 4.01 SP2 or later must be installed and operational.
To install TS2Mascot, run the
Unless 4000 Series Explorer or GPS Explorer is already installed
on the target PC, you may need to create a local Oracle service name,
as described in the setup
TS2Mascot is very simple to use. It is launched from the Windows Start menu, (Programs; Mascot; TS2Mascot).
The folder tree in the Spot Sets frame is populated with project folders from the 4000 series database.
If you have more than one 4000 series database, choose Change to connect to a different database.
Navigate the folder tree and select a spot set icon. This will populate the adjacent drop down list
with the job runs for the spot set. The grid area displays details of individual job run items
for the selected job run.
Peak filtering settings are equivalent to those used in AB SCIEX GPS Explorer.
The mass range limits only apply to MS/MS fragment ion peaks.
The contents of the Peak Filtering and Mascot Server URL fields are sticky. If you change them,
then process a spot set, the new values will be remembered.
Save peak list invokes a standard file selection dialog before processing the data.
Before choosing Mascot Search, ensure that you have entered a valid URL for a Mascot
server. You can enter the URL of the Matrix Science public web site, but remember that
this has a limit of 300 spectra in a single search.
While the peak list is being created, a progress bar is displayed, and all the
controls are disabled apart from a Cancel button. Once peak list export is complete,
if you have chosen Mascot Search, the default web browser will be launched and the
Mascot MS/MS search form displayed. The search title and data file path are filled in
automatically. The other settings are the search form defaults, (which can be customised
by following a link on the search form selection page).
If you want to perform iTRAQ quantitation outside of GPS Explorer, it is important to use TS2Mascot
because the peak areas in the peak list exported by Peaks to Mascot or submitted to a Mascot server by GPS
Explorer are not quite the same as those required for quantitation. For a detailed description of Mascot
support for iTRAQ quantitation, refer to the quantitation help page for the
Automation using Mascot Daemon
Mascot Daemon can use TS2Mascot as a data import filter. First create a suitable parameter
set and save it. If the parameter set is for searching MS/MS data, the file format should be specified as
In the Task editor, if you want to use TS2Mascot as the data import filter, select the parameter
set and then choose the AB 4000 Series TS2Mascot option from the data import filters.
The Add Files ... button invokes a database browser window.
Choose Export all MS/MS spectra in job run for a single search of all the MS/MS spectra in the job run.
Any row selection in the data grid has no effect when this option is used.
Alternatively, choose Export selected spectra only and select one or more rows
in the data grid to search these spectra as separate searches, (e.g. a batch of PMFs).
This option works for both MS and MS/MS data.
The MS Data Converter is a free download from AB Sciex.
It supports both Wiff files and TOF/TOF data stored in an Oracle database. Output can be a simple MGF peak list
or an mzML file containing a fairly complete representation of the raw data.
Converting a TOF/TOF spot set into an mzML file enables Mascot Distiller to be used for quantitation. Select
the MS and MS/MS job runs corresponding to a single LC separation. If you have multiple separations,
corresponding to fractions or replicates, each one must be exported to a separate mzML file, and you can then
create a Distiller multi-file project for the set of files. The MS Data Converter
User Guide explains how to format a path to specify the data to be exported.
In most cases, you should choose to export profile data. If you intend to open the file in Distiller,
you need to add an undocumented switch to the command line,
/zeropadding, to preserve data points with zero intensity. For example
AB_SCIEX_MS_Converter.exe TOFTOF "4800\Polka\RASH\SILAC
experiment 20110617 X5\3\2" -profile MZML C:\tmp\silac_20110617_x5.mzml /zeropadding
If you do not do this, you may observe artefacts in weak spectra that compromise peak picking.
The increase in size of the mzML file when the /zeropadding flag is used is much less than with
Wiff files because there is normally some baseline offset in TOF/TOF data, so that the signal rarely
drops to zero.
Once your data are in mzML format, Mascot
Distiller can be used quantitation. Suitable Distiller processing options for profile and centroided
MALDI data exported to mzML are here:
If Distiller includes the optional Mascot Daemon Toolbox, Mascot Daemon
can be used to automate peak picking, Mascot search, quantitation,
and saving the Distiller project file. This screen shot shows an example of a SILAC experiment using
data acquired on a 4800, courtesy of Ida Chiara Guerrera and Francois Guillonneau, Universite Paris Descartes.