Fragment ion XIC intensity

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Fragment ion XIC intensity stephen swatkoski  2019-04-05
 

Hi,

Thank you for the great support you provide. Your responses to my previous posts have been very helpful. I hope this is the last question I have for awhile. I am analyzing PRM data and noticed that when I click on a point along a fragment ion XIC the intensity of the fragment is different than the intensity of the fragment ion in the raw ms/ms data. This is observed consistently across multiple peptides and fragments of those peptides. The XIC intensity at a given point along the curve always appears to be higher that the fragment ion displayed in the ms/ms spectrum. Can you explain why this is the case? Please see attached example. The intensity at the apex of the XIC for y10 is about 2.7e6. However, the intensity of y10 in the raw ms/ms spectrum at that same time point is lower (7.2e5).

Thank you,
Steve

 
 
Brendan MacLean responded:  2019-04-05

Probably, if you zoom in, you will find that the peaks you are seeing in your spectra are in profile mode and not centroided, which means that each filter will contain more than a single peak. The value you see in the chromatograms is the sum of the peaks inside your extraction filter. This is generally a more precise measure than the height of a profile peak, which is what you are seeing when you are zoomed out like in your screenshot. Press the button with the magnifying glass icon to zoom in and you will see that the colored extraction range contains more than a single peak.

If you use the "Centroided" mass analyzer type in your Transition Settings - Full-Scan tab, then the relationship will generally look more like what you are expecting, where most of the time the extraction range will contain just one centroided peak.

This page and the next from Skyline Tutorial Webinar #14 shows it well on SCIEX 5600 spectra both profile and centroided:

https://skyline.ms/_webdav/home/software/Skyline/events/2017 Webinars/Webinar 14/%40files/Webinar 14 (MacLean).pdf#page=18

Hope this helps to clarify. If you feel I am wrong, send a screenshot of your settings using Centroided for the mass analyzer and a zoomed in extraction window like the ones in the slides I have pointed you to.

Thanks for posting to the support board and including a screenshot.

--Brendan