Collision Energy Optimisation on Agilent Ultivo QQQ

Collision Energy Optimisation on Agilent Ultivo QQQ a grayson  2021-04-12


I have been using an Agilent Ultivo triple quadrupole instrument to perform MRM analysis of peptides. I used Skyline to create a method which optimises the collision energy of 5 transitions for each peptide precursor. In Settings - Transition Settings, I selected Agilent QQQ from the dropdown menu for collision energy, which made a transition list with collision energy step size 3V and step count 3.

Upon running this method on the Ultivo and importing the data into Skyline, some transitions only show 5 or 6 out of 7 collision energy results, and the Retention Time and Peak Area - Replicate Comparisson graphs will only show a select few of these results. For example, in the screenshot attached, the transition selected shows data for 6 different collision energies was recorded (not 7, why?) and the graphs for Retention Time and Peak Area on the bottom only plot four of these.

I have double checked the method I ran and confirmed all seven collision energies were meant to be analysed for all transitions. Could you please advise why I cannot see all 7 collision energies for some of the transitions, but can for others? There does not seem to be a pattern in which transitions it did not record all data for.

Kind Regards,

Brendan MacLean responded:  2021-04-12

Hi Amy,
Can you provide your file using File > Share with "Minimize libraries"? It should be small enough to just attach to this support request, but if not, you can post it to:

That does look a little strange to me. Thanks for posting the screenshot.


a grayson responded:  2021-04-13

Hi Brendan,

I have uploaded a .zip file containing my raw data collected on the Ultivo, and the Skyline file I used to analyse the data, into the file sharing folder via the link you provided.

If you require any more information or files, please let me know. Thank you for your help regarding this.

Kind Regards,

a grayson responded:  2021-05-13

Hi Brendan,

Just wondered if there was any update on solving this issue?

Best Wishes,

Nick Shulman responded:  2021-05-13

The chromatograms in "INJ2_CE.d" inside of "Skyline Collision Energy Ultivo" do not look like they came from a collision energy optimization method that Skyline would have created.

When Skyline exports a collision energy optimization method, for each transition, Skyline adds a set of chromatograms whose Q1 values are all the same, and whose Q3 values differ by 0.01. So, if the precursor/product mz's were "425.2451" and "403.23", and the step count was 3, I would expect to see the following chromatograms:
Q1=425.2451 Q3=403.20
Q1=425.2451 Q3=403.21
Q1=425.2451 Q3=403.22
Q1=425.2451 Q3=403.23
Q1=425.2451 Q3=403.24
Q1=425.2451 Q3=403.25
Q1=425.2451 Q3=403.26

Instead, what I see in there are a group of chromatograms with the Q1 value 425.2. There are six chromatograms whose Q3 is 403.196 and one chromatogram Q3=403.296 (and there are other chromatograms that correspond to different transitions for the same precursor).

The reason that Skyline needs to see chromatograms whose Q3 values are 0.01 apart from each other is that Skyline does not know how to read the collision energy from the raw file. If Skyline sees a set of chromatograms with exactly the same Q1 and Q3 values, Skyline does not have any way to know which chromatogram is supposed to correspond to which collision energy.

Was this data collected using a method that Skyline had created? Do you have any idea why the Q3 values would not be what Skyline is expecting?
-- Nick