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Skyline is missing some MRM chromatograms and spectra in my Sciex MIDAS files

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Skyline is missing some MRM chromatograms and spectra in my Sciex MIDAS files vonroep  2024-06-04 09:30
 

Hallo Skyline Team,
I recently started using Skyline to investigate my MIDAS runs (MRM triggers MSMS) . I started with simple old experiments and want to change over to complex naturally crosslinked proteins. However already in the simple runs I find discrepancies between Skyline and my Analyst software. I guess some of my parameters are not set appropriately. However after 4h of tutorial reading and testing I am still stuck with the missing MRMs. I attached a document with the screen shots of Analyst and Skyline and I also attached the Skyline experiment.

Thanks in advance for your time and help.
Kind regards

Edda von Roepenack-Lahaye
 
 
Nick Shulman responded:  2024-06-04 09:52
Can you send us your .wiff file and the .wiff.scan file that goes with it?
That would be:
ADC mix digest MRM IDA fix 20min 02122021 b.wiff
and
ADC mix digest MRM IDA fix 20min 02122021 b.wiff.scan

The best way to send a Skyline document is to use the "File > Share" menu item to create a .sky.zip file.

The .sky.zip file will contain the .sky and .skyd files that you already attached, but it will also contain other supporting files including the .midas file which Skyline created while extracting chromatograms.
The "Share Document" dialog also gives you the option to include the raw (.wiff) files.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick
 
vonroep responded:  2024-06-04 10:53
Hi Nick,
I attached the zip file. I also choose the results file in the dialogue, which should be the midas run.

Thanks a lot.

Edda
 
Nick Shulman responded:  2024-06-04 16:36
Thank you for sending that .wiff and .wiff.scan file.
I see that you have several chromatograms that look like they belong together except that the Q1 values are slightly different.
For instance, there is a chromatogram with Q1=575.3 Q3=832.5 and another one with Q1=575.31 Q3=731.4
Whenever there is even a slight difference in the Q1 value, Skyline assumes that the chromatograms are intended for different molecules.

When Skyline is trying to decide which chromatograms correspond to which precursors and transitions in your Skyline document, Skyline first groups the chromatograms by Q1 value.
Then, for each precursor in your Skyline document, Skyline examines each group where the Q1 value is close in value to the precursor m/z and Skyline counts the number of transitions whose product m/z is close in value to the Q3 value of a chromatogram in the group.
The closeness of matching that is required between the m/z and Q1/Q3 value is determined by the "Method match tolerance m/z" value at "Settings > Transition Settings > Instrument".

-- Nick
 
vonroep responded:  2024-06-05 01:50
Good morning,
Thanks for the quick reply. I tested different “Method match tolerance” settings from 0,05 til 0,6 but it did not influence these particular transition pairs, with the 0,01Da differences in the Q1. I guess Skyline cannot match two nearly identical Q1s with one particular calculated precursor and chooses only one of them in the end. Or is there another parameter, which influences the Q1 picking? I also tested the resolution settings in the Transition Settings but to no avail.

Kind regards
Edda