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:
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.
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.