Table of Contents

guest
2023-02-07
   Vendor-Specific Instrument Tuning Parameters
     Support for Bruker TOF Instruments
     SCIEX Instrument Settings
     Export SRM Methods for a Thermo LTQ

Vendor-Specific Instrument Tuning Parameters


As of release 3.5, Skyline's small molecule support includes the ability to explicitly set many vendor-specific instrument tuning parameters on a per-precursor basis.

The "Insert Transition List" dialog for small molecules now has columns for importing various vendor-specific values such as "S-Lens", "Cone Voltage", "Declustering Potential" and "Compensation Voltage", along with the previously implemented ability to explicitly set "Collision Energy", "Retention Time" "Retention Time Window", "Drift Time", and "Drift Time High Energy Offset". These values can also be modified in the Document Grid.

These values can also be modified in the document grid for peptides (formerly this was only possible for small molecules).

By default S-Lens values are not written: a new checkbox in the Export Method dialog enables this for appropriate Thermo outputs. On the commandline side, there is a new argument "exp-use-s-lens" for this.




Support for Bruker TOF Instruments


Skyline v2.1 introduces fully integrated support for Bruker micrOTOF-Q and maXis series instruments.  The Skyline support for working with full-scan mass spectra has been extended to Bruker TOF instruments, and data acquired with them in several modes:

  • MS1 Filtering - chromatogram extraction from MS1 scans in data dependent acquisition (DDA) experiments
  • Targeted MS/MS (PRM) - chromatogram extraction from MS/MS scans in pseudo-SRM experiments
  • Data Independent Acquisition (DIA) - chromatogram extraction from MS/MS scans acquired in various isolation schemes
    • Consecutive wide windows
    • All Ions - alternating low energy and high energy scans of the entire instrument range

For more information on working with Skyline and Bruker TOF instruments, consult the following resources:

  • HUPO 2012 Bruker Lunch Seminar [PowerPoint][PDF]
  • Targeted Proteomics with Bruker TOF Instruments tutorial [PDF]

The following supporting files may also be useful:

  • Skyline template documents for Bruker TOF data analysis [ZIP]
  • Script for adding retention times to MGF files for peptide searches [Script]



SCIEX Instrument Settings


Attached to this page you will find Skyline settings files created by SCIEX as helpful defaults for the QTRAP and TripleTOF instruments.

To load these settings files into Skyline, perform the following steps:

  • On the Settings menu, click Import.
  • Select one of the .skys files.
  • Click the Open button.

This will add a new menu item to the Skyline Settings menu, either QQQ_QTRAP_Environment or TripleTOF_Environment depending on which file you imported.  To change the settings on your current document, simply choose one of these menu items.  This will change the document settings to the defaults that AB SCIEX has created for their instruments.

 

Note that if you employ explicit tuning parameters such as Explicit Collision Energy or Explicit Declustering Potential these must be absolute values (i.e. positive numbers). When writing transition lists for negative ion mode operation, Skyline will automatically express them as negative values.




Export SRM Methods for a Thermo LTQ


You may know that Skyline documents can be exported to MRM/SRM transition lists for all of the major triple quadrupole instruments available today.  You may even know that Skyline documents can be exported directly to native methods for some of these instruments.  But, Skyline can also export SRM method files for the Thermo-Scientific LTQ.

An ion trap instrument like the LTQ may not have the sensitivity of a triple quadrupole, but you can still use one for targeted proteomics, and you can use SRM on the LTQ as a quality control measure for your liquid chromatography.

While you can export an existing Skyline document to a native LTQ method for SRM, you should be aware of a couple settings before you do.  To prepare your document for use with the LTQ, perform the following steps:

  • On the Settings menu, click Transition Settings.
  • Click the Instrument tab.
  • Check Dynamic min product m/z.
  • Enter the correct value (e.g. 2000) for your instrument into the Max m/z field.
  • Click the OK button.

The first setting will restrict the product m/z values Skyline will allow to being greater than a dynamic minimum, based on the precursor m/z, consistent with the limits the LTQ imposes.  The second setting will restrict both the precursor and product m/z values Skyline allows to be consistent with what your LTQ is calibrated to allow.

If you have done this on an existing document, you should probably review your transitions to be sure Skyline has not removed anything important.  Small product ions may no longer be measurable on the LTQ, which could cause some precursors to contain fewer transitions than you want for your experiment.

If this is a new document, you can now enter the peptides you are interested in targeting as you would normally, understanding that some smaller product ions that you would normally see will no longer be available in the Skyline user interface.

When you are ready to export a LTQ method file for your Skyline document, you must transfer your Skyline document to the instrument control computer for your LTQ instrument, where you will also need to have Skyline installed.  If you are using a complex document involving spectral libraries, you may want to consider using the Share command on the File menu, as described in the tip on Sharing Skyline Documents in Manuscripts.

Once you have your Skyline document open on the LTQ instrument control computer, you are ready to export it to a native LTQ method or .meth file.  To do this, preform the following steps:

  • On the File menu, choose Export, and click Method.
  • Choose 'Thermo LTQ' from the Instrument type list.
  • Click the Browse button beside the Template file field.
  • Browse to a .meth file you will use as a template for all settings other than the m/z values to monitor.
  • Click the Open button in the Method Template form.
  • Choose options in the rest of the Export Method form as you would for exporting a transition list or a TSQ native method.
  • Click the OK button.
  • Enter a name for your method, or root name if creating multiple methods.
  • Click the Save button.
This will create a LTQ method or multiple methods which you can use to make SRM measurements on your LTQ.  Once these are complete, you can import the resulting Thermo .RAW files as you would .RAW files from a TSQ triple quadrupole instrument.

Using SRM for Liquid Chromotography Quality Control on the LTQ

Issues with chromatography can easily go unnoticed on systems performing predominantly shotgun data dependent analysis (DDA).  They can, however, still greatly effect performance, especially if you are hoping to use tools that analyze MS1 scan data for quantification and feature detection.  At the MacCoss lab, we are using SRM methods generated with Skyline to monitor LTQ system performance.  Every tenth run on our LTQ instruments, we inject a known standard mix and measure its abundant peptides using SRM.  We find that measured retention times and peak shapes of known peptides give us increased visibility into system performance of the LTQ.

At present we are injecting the "6 Bovine Tryptic Digest Equal Molar Mix PTD/00001/63" from Michrom Bioresources, Inc., running SRM methods generated with this Skyline document:

Bovine_Mix_QC.sky

Below are examples of Skyline displaying both failing and passing runs on our LTQ Velos. Each QC replicate displayed in Skyline was taken as every tenth injection with the other 9 injections used for normal shotgun MS/MS measurement.

Failing:
In the QC runs shown below, chromatography issues first appear between runs 9 and 12. By QC13, the system is clearly not functioning acceptably.

Passing: In the 33 QC runs shown below, both a retention time drift of about 2 minutes and decreasing intensity are visible, but measurements remain within an acceptable range throughout.