Hi Julie,

Can you provide the Skyline document that you're using? Please do so with Skyline's File>Share>Complete menu item.

Thanks for using the Skyline support board,

Brian Pratt

Certainly very strange. I cannot reproduce this from scratch myself. We are definitely going to need your file. Is this the same exact file opened in the two different versions? It may be in how the ions get persisted.

Thanks for posting this, and we look forward to seeing your .sky.zip file.

Skyline already supports extraction from MS3. The MS/MS filtering actually applies to MS(N) filtering, as long as the MS1 isolation matches your precursor. So, what we don't yet really support is when you are producing different MS3 spectra from the same peptide precursor but different MS/MS fragments. As long as each MS3 has a distinct MS1-level peptide precursor m/z, your PRM with MS3 spectra should work just fine.

--Brendan

Hi Brian and Brendan,

thanks a lot for the quick replies!

It is not the same file in the two versions. Version 4.2 couldn't open the file from version 20, so I created a file from scratch there. Yet today I tried the reverse, and I could open the file from v4.2 in v20.2 and it had the TMT ions there, so at least I have a workaround for now :)

I am attaching my v20.2 sky.zip file here, as that is the one that's troublesome, while v4.2 seems to be working fine.

Interesting news about the MS3 extraction! I am not sure I fully understand your comment about the MS1 isolation matching the precursor... Does that mean that the isolations for MS2 and MS3 fragmentation need to target the same mass? We typically run multinotch isolation of multiple MS2 fragments for MS3... I will certainly look in more detail at the MS/MS filtering to find out more. Thanks!

Thanks a lot for your help and insights! I really appreciate it!
Best
Julie

Hi Julie,

Thanks for providing the file. It would probably be useful for me to see the v4.2 file as well, if you wouldn't mind.

Thanks!

Brian

Hi Julie,
For MS3 we have tested exactly what you are describing with Phil Remes of Thermo, and it worked. I am saying that Skyline essentially ignores the MS2-level isolation information. MS3 has 2 levels of isolation information, the MS1 level - the peptide precursor m/z, and the MS2 level - the fragment ion or ions that get isolated from the first time in the collision cell. These are recorded in the raw data along with the final resulting spectrum. Skyline will extract product ions from essentially any level of spectrum MS2, MS3, etc. as long as its MS1 precursor m/z matches, which works great as long as all of the spectra collected in your raw data for a specific precursor m/z are for the same precursor target. It would not work so well if you were collecting both MS2 and MS3 spectra for the same precursor m/z or you were collecting MS3 spectra for the same precursor m/z value which were differentiated by their MS2 isolation. Both of these turn out to be relatively infrequent, and neither occurred in Phil's test data.

As for the mystery of the missing TMT ions, that is due to the Transition Settings - Instrument - Min m/z setting being 250. Essentially, you have told Skyline that your instrument is not capable of measuring these ions. If you lower this value to 100 m/z, then the TMT ions will be accessible to you. You must have a lower value in the document you created with Skyline 4.2. If you were trying to use this minimum to limit the size of the y- and b-ions that you will accept, then you should instead increase your ion range to something like "ion 3" (i.e. excluding y1, y2, b1, b2) instead of using "ion 1" which allows even y1.

One setting you have which has been a longtime confusion since Andrew Stergachis in our lab used it for an early paper, and that is limiting your ion range to "last ion - 1". Andrew mistook this to mean what he was calling in his paper y(n - 1), where n was the length of the peptide. However, it actually means y(n - 2). The option "last ion" is not referring to the precursor itself. That is, when you have a 10 amino acid peptid, its "last ion" will be y9 or b9. Whereas, its "last ion - 1" will be y8 or b8. This has been confusing enough for Skyline users that I am strongly considering removing the "last ion - n" options and warning if a user tries to click the OK button in a Transition Settings form where a value like this has been brought forward through backward compatibility with an old document.

Were you really intending to exclude the longest possible y- and b-ions and if so, why exactly?

Hope this gets you past your immediate issue, which is not a version related issue. Thanks for supplying your document and working with us on the Skyline support board.

--Brendan

Hi Brendan and Brian,

thank you so much for the super comprehensive reply! I wouldn't even have considered the transition min m/z setting, but yes, it makes total sense! This has immediately solved my problem! Thank you so much!

Also thanks for the explanation regarding MS3, that's certainly something I will try very soon!
The product ion settings are not my standard settings, I just tried to be the least restrictive when trying to get the TMT ions to show up. I typically exclude the b1 and y1 from the product list, but I didn't know about the "last ion -1" thing! That's great to know and I will certainly change my standard settings to "last ion" and make my colleagues aware of this as well.

Again, thank you so much for developing this awesome software and for actively supporting the users!
Have a great weekend!
Best
Julie