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Applied Biosystems | MDS Sciex Analyst

mascot.dll

The latest version of the Mascot script for Analyst, which provides a convenient interface to Mascot, can be downloaded here:

Before installing mascot.dll, you should verify that Analyst has the latest hot fixes applied.

There are some troubleshooting tips below.

Note that the screen shots shown below are from the 1.6b21 version of the script, and there may be minor differences in later versions.

Interactive Searching

When used interactively, the Mascot script performs data reduction of MS and MS/MS data, creating a Mascot Generic Format (MGF) peak list, and sending it to a Mascot search form. The Mascot server can be either the public web site or an in-house server.

Full details of the dialogs and options can be found in mascot.doc, which is copied to the Analyst help directory during installation. Briefly, the available options depend on the contents of the active Analyst pane at the time the script is invoked. (NB The display must be the standard 'TIC' display, not IDA Explorer).

mascot.dll 1

  • The current spectrum If the active Analyst pane is a centroided spectrum, (either MS or MS/MS), this option can be used to search just that spectrum
  • All MS-MS spectra in the file This option can be used to perform time domain processing of all the MS/MS spectra from a single sample. That is, MS/MS spectra from the same precursor, eluting within a specified time interval, will be summed together
  • All MS-MS spectra from selected region in TIC If the active Analyst pane is a TIC that contains a selected region, this option can be used to perform time domain processing over just the selected region. This can speed up the processing time if only a portion of the run is known to contain useful data
  • All MS-MS spectra from all samples This option can be used to perform time domain processing of all the MS/MS spectra for all samples in a multi-sample WIFF file
Clicking on the Options... button displays the processing options for data reduction. Tooltip help is provided for most options.

mascot.dll 2

The 'Set default parameters' button displays Mascot 'Set Defaults' form , allowing you to define default search form settings.

Note:If you are processing iTRAQtm data, then you should clear the 'De-isotope MS/MS data' checkbox, since this may distort the iTRAQ reporter ion peaks.

Automation using Mascot Daemon

Mascot Daemon can use mascot.dll as a data import filter, eliminating the need to create peak lists from WIFF files manually. To use this facility, first create a suitable parameter set and save it. The Data format should be specified as Mascot Generic.

daemon parameter tab

In the Task editor, Choose the 'AB | MDS Sciex Analyst .WIFF file' option from the data import filters. This option will be available if mascot.dll is installed and registered on the PC running Mascot Daemon.

daemon task tab

Clicking on the Options button will invoke a dialog that is very similar to the interactive mascot.dll dialog. If a WIFF file contains multiple samples, you must choose whether to merge the data from all samples into a single Mascot search or generate a separate search for each sample. Some recent mascot.dll options may not be represented in the Daemon dialog. In such cases, the setting for the missing option will be the same as when the script was last used interactively, from Analyst. An example would be the 'De-isotope MS/MS data' checkbox.

daemon options

Press OK to save the options, and then continue to set up either a batch or real-time monitor task. Note that when the task is run, the options settings are saved as defaults for next time, but each task can have different options settings.

Note: The facility to have multiple samples in a single WIFF file, and merge them all together for searching, is ideal for Mudpit. Each sample would then represent the LC-MS/MS data from a fraction bumped off the strong cation exchange column.

Real-time Monitor Mode

One type of Daemon task is a batch task, where a list of WIFF files is searched immediately, or at some pre-set time. Alternatively, it can be a real-time monitor task, monitoring a specified path for new WIFF files. The advantage of a real-time monitor task is that data files can be submitted to Mascot immediately after acquisition and without any user intervention.

daemon task tab

In real-time monitor mode, it is important that Mascot Daemon waits until a file is complete before submitting it to Mascot. To avoid taking a file that is still being written, it checks the file size at intervals, and waits until it has stopped increasing. The default interval is 60 seconds, which works fine in most cases, but may not be long enough if there are periods when the WIFF file size grows only slowly. If you observe that Daemon is submitting a search before the WIFF file is complete, increase the interval by going to the Timer Settings tab of the Preferences dialog. Increase the value of 'Delay after failing to open read-locked file' from 60 seconds to (say) 600 seconds.

daemon preferences

Troubleshooting

In order to import WIFF files in Mascot Daemon, both Analyst and mascot.dll must be installed on the same PC as Daemon. If you still don't see the 'AB | MDS Sciex Analyst .WIFF file' option on the data import filter drop-down list, or if you see the following error message, then you either have an old version of mascot.dll, or it is not installed and registered correctly.

Run-time error '430': Class does not support automation or does not support expected interface

The fix is to download and install the latest version of mascot.dll.

If you get the message:

Cannot load driver library FMWIFFCompDocNTDriver.dll Make sure the DLL is in your path or in the same directory as your .exe <C:\program files\daemon\daemon.exe>
The problem is that the Analyst\bin directory is not on your path. Go to the System applet in the Control panel and choose the Advanced tab. Select Environment Variables, and add the full path to the Analyst\bin directory to the end of the path. You may have to re-boot the system for the change to take effect.

system applet

Acknowledgements

mascot.dll was a joint development between Applied Biosystems | MDS Sciex and Matrix Science
 
 
Copyright © 2010 Matrix Science Ltd. All Rights Reserved.