Different results from raw data that are nearly the same in MassLynx

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Different results from raw data that are nearly the same in MassLynx anna walke  2021-01-07
 

Dear SkyLine Team,

I'm working with a simple method on a waters Premier Pro QToF to weekly check system performance by analyzing a standard of angiotensin II. Measurement is performed by Full Scan (function 1) and MS/MS scan (function 2) in parallel over the entire time range of the method.

Unfortunately when analyzing my raw data with Skyline (version 20.2.0.343) I get very different results compared to manually analyzing the same data in MassLynx. I added screenshots of this and also attached the corresponding information of my LC-MS experiment. Within the experiment report I highlighted the different paramters in yellow, but there are only little differences.

I do not understand why the same SkyLine Document (also attached below) with same molecule and transition settings gives that different results after integration.

Could you please help me or give a hint towards a possible solution?

With kind regards,
Anna

 
 
Nick Shulman responded:  2021-01-07
Anna,

Can you send us your Waters .raw file directories ("AGII Std 16.raw" and "AGII Std 01.raw")?
You can zip up those directories and upload them here:
https://skyline.ms/files.url

When Skyline is extracting chromatograms from MS1 and MS2 scans, Skyline adds all of the intensities across a m/z channel centered on the m/z of the ion of interest. The width of the channel that Skyline sums across is determined by the resolution settings that you have specified at "Settings > Transition Settings > Full Scan". One thing that might be causing a difference between the numbers that Skyline and MassLynx gets would be if the bulk of the intensity was near the edge of the channel that Skyline was summing across.

If you click on the chromatogram graph, you can get Skyline to show you the spectrum from the raw file, and you can see how well the intensity is centered on the m/z channel.

-- Nick
 
anna walke responded:  2021-01-07
Dear Nick,

thanks for your information. I uploaded the requested files and will check the intensities you mentioned.

Anna
 
Nick Shulman responded:  2021-01-07
Thank you for sending those files.

When I click on the chromatograms for "Std 16", I can see that Skyline is extracting chromatograms across a small range at the uppermost edge of the m/z range of the peak in the spectrum (see attached pictures).

One thing that you could do to correct this problem is to go to:
Settings > Transition Settings > Full Scan
and change the "Resolving Power" in both the "MS1 filtering" and "MS/MS filtering" sections to a smaller number such as "10000". (you current have it set to 30000).
After you make that change you will need to tell Skyline to extract the chromatograms again (Edit > Manage Results > Reimport) in order to actually see the change take effect.

If you change the resolution to 10000, then Skyline will sum intensities over a wider m/z channel, and it will covering much more of the peak that is in that spectrum.

It is also possible that you mass spectrometer needed to have been calibrated differently. The m/z channel that Skyline was summing across was much better centered on the spectrum peak in your "Std 01" file. I imagine that calibrating Waters instruments has something to do with "lockmass correction". I do not know anything about how to do lockmass correction, but I imagine other people on this support board would be able to answer questions you might have on that topic.
-- Nick