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Agilent QTOF targeted method optimisation

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Agilent QTOF targeted method optimisation reka haraszi  2018-04-19 01:35
 
I know it is not very common to do targeted on a QTOF but... I did a set of peptide transitions with the help of the latest version of Skyline and exported the transition list for Agilent QTOF, see attached as glutenCTR2QTOF.csv. What I do not understand is what are the 0.01 m/z increments in the product ion mass associated with an increase of 3 in CE? The optimisation setting I put was for transitions (not precursors). When I set it for precursor, it gave me only one CE value (attached as glutenCTR2QTOF_pre.csv). Thanks, Reka
 
 
Brendan MacLean responded:  2018-04-19 10:31
So, you are trying to perform CE optimization on a QTOF?

For reasons which should be fairly obvious, I don't think it is possible to optimize CE by transition on a QTOF, since transitions are not measured separately but together in a single scan.

I would recommend reviewing the PRM tutorial:

http://skyline.ms/tutorial_targeted_msms.url

And any of the PRM webinars:

http://skyline.ms/webinar03.url
http://skyline.ms/webinar09.url
http://skyline.ms/webinar17.url

For PRM, we would expect you to use File > Export > Isolation List, or your own custom report as in webinar 17 or page 13 of the tutorial.

I am not really clear on what you are hoping to achieve with a transition list for a triple-quadrupole on your QTOF.

Thanks for posting to the Skyline support board.

--Brendan
 
reka haraszi responded:  2018-04-20 03:05
Thank you, Brendan. To provide a bit more context, I selected some MRMs to screen for gluten peptides and did some targeted runs on an Agilent QQQ using CE optimisation in Skyline for QQQ. The results were not convincing, weak signal responses and questionable identifications obtained from materials where high response was expected. There maybe a problem with the sample prep but let's assume it was good enough to show at least some response especially because most of the MRMs were published. To speed things up I thought I acquire an MS profile on an LC-QTOF to check the presence of some of the parent ions I targeted on the QQQ and yes, I could find some of them but obviously had no idea about the fragmentation, so the next step was to do targeted MSMS run on the LC-QTOF to check what transitions can be detected for these parent ions. This is when I created the CE optimisation files for QTOF hoping to copy paste the target masses and related settings into the instrument software. Then I noticed the 0.01m/z for each 3 CE per each parent mass and asked the question what this is. Meanwhile, it became clear that the format generated by Skyline is actually not matching (now I understand why) and also it is only possible to target for the parents and not specific parent-product pairs. I will look into the PRM tutorials as you suggested.