PRM Quantification of Croos-linked Peptides

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PRM Quantification of Croos-linked Peptides julius fuersch  2019-09-09
 

Hi,
I am doing quantification of cross-links in PRM mode. I generated my transition list by the plugin Xlink Transition Calculator. I am using the latest Skyline version. I recognized that I need to increase the ion match tolerance up to 0.5 m/z in the transition settings to get matched peptides. Nevertheless, after doing this, the upshowing best matched peptides have very low mass error (0.5-10 ppm, measurement was on an Oribitrap Fusion). As a consequence there are a lot of other matching peptides, often difficult to judge which one is the correct hit.
Additionally when using this plugin, it seems not to be possible to add decoys because skyline runs in the molecule mode. Changing to proteomics mode is not possible! So I can not use mProphet.

Do you have any suggestions how I can improve my pick picking? I know approximately the expected retention time, so now I am only trusting hits within a certain time range. Do you have any ideas to make a reliable pick picking?

In the PP file, I tried to illustrate the problem of more than one well matching peak with a red box and the other mentioned points!!

Thanks a lot

 
 
Nick Shulman responded:  2019-09-09
In general, if you have a small molecule document and you want to have access to the proteomics menu item, you should switch to "Mixed" mode instead of "Proteomics" mode.

However, that will not help you in terms of adding decoy peptides.

I am not sure that I understand why you had to increase the Method Match Tolerance M/Z to 0.5. That number controls how close the isolation window of an MS2 scan has to be to the calculated m/z of the precursor in order for Skyline to decide that that MS2 scan belongs to that precursor.
If you have to set that to a big number like 0.5, that means that the thing that generated your method had a very different idea of what these molecules were than what Skyline thinks.
Do you have any idea why that would be?

It might be helpful if you send us your Skyline document and at least one of your .raw files.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

Also, send us at least one of your .raw files.
-- Nick
 
julius fuersch responded:  2019-09-10
Hi Nick,
thanks a lot. This was already extremely helpful. I think I need to write an bug report to the developer of the plugin, because the plugin is responsible for the switching to the molecule mode, however we are working with cross-linked peptides. If you can not add decoys, mProphet is not possible which is a problem!
I think I also understood now why I need to increase the match tolerance. I recognized that the plugin always calculates the precursor m/z (value written below the peptide sequence in the transition list) exactly one m/z unit different from the precursor m/z that is written in the pull down menu (if you press the "+" to open the fragment masses). But the precursor m/z within the pull down menu is the correct one!! I tested this also for an "test transition list" which is given by the plugin developer, here this is also the case. I sent you the skyline document for you to ckeck by your self, but I am pretty sure by now.
Thanks a lot
Julius
 
julius fuersch responded:  2019-09-10
Hi Nick,
I uploaded the skyline document FUS_salt_gradient_PRM_final_file.sky.
One raw file is attached
 
Brian Pratt responded:  2019-09-10
Hi Julius,

It should be noted that the switch to molecule mode isn't a bug per se - Skyline doesn't have any way to specifically model crosslinked peptides, so the plugin presents them to Skyline as generalized molecules defined by chemical formula rather than by amino acid sequence. As such, much of what Skyline can do with actual peptides (predict fragmentation, generate decoys etc) is no longer applicable and so Skyline doesn't present those peptide-specific parts of the UI. It's not the plugin developer doing that UI change, it's Skyline itself.

Best Regards,

Brian Pratt
 
julius fuersch responded:  2019-09-11
Hi Brian, Hi Nick,

thanks a lot for your answers. It was extremely helpful. I also talked to the plugin developer who explained to me that they had to use the molecule interface to make Crosslink detection possible in Skyline. He also explained that the plugin always calculates the mass of the most abundant isotope as overall peptide mass and the monoisotopic precursor masses are listed in the drop down menu. Unfortunetly I generated my inclusion list for the PRM measurement based on the monoisotopic masses which I got from another cross-link specific search pipeline. Now I have the problem that the peptide masses calculated by the plugin (most abundant isotope) do not match excactly the masses within my inclusion list, I could work around it by setting the method match tolerance to the maximum of 0.6 m/z, but there are still some Peptides cannot be matched by Skyline because the mass difference is bigger than 0.6 m/z. Addittionally if I have Peptides with very similiar m/z, I will have mismatching. Do you see any solution despite of reameasuring the samples with the mass calculated by the plugin. Is it possible to tell Skyline to pick the monoisotopic mass for matching?

Thanks a lot

Julius
 
Brian Pratt responded:  2019-09-11
>> Is it possible to tell Skyline to pick the monoisotopic mass for matching?
I think you are asking about precursor isotope selection in Skyline? It appears that the plugin only provides mass values, and a full chemical formula is needed for Skyline to perform isotope calculations. So no, there's no way for Skyline to adjust that automatically.

Perhaps the best move would be to see about adjusting the plugin's source code to emit the monoisotopic values if that's what you really want to be monitoring. Or you could carefully hack the existing .sky file to use those values instead, then reimport your replicates. (Not sure how that would go with the fragments though.)

Best,

Brian
 
engj responded:  2019-09-12
Regarding monoisotopic masses, the documentation for the cross-link transition calculator plug-in describes how you can direct the plug-in to report monoisotopic m/z's by adding "MONO" to the cross-link masses line. You can find this on page 4 of "XLinkPlugin.pdf".
 
julius fuersch responded:  2019-09-13
Thanks a lot guys, adding MONO was the crucial hint. Now, everything works perfectly