Pointed peaks vmohanty  2024-08-23 08:28
 

Hi!

I am trying to optimize my PRM method on Exploris 120 coupled to Vanquish Neo.

I am getting pointed transition peaks on skyline for few peptides for some LC-MS methods. I have attached the pics. Could you explain the reason for pointed peaks and can it be considered as good identification?

I have attached 5 pics. However, I know Pic4 right side seems good. But I am unsure what causing pointed peaks in remaining pics.

VM

 
 
Nick Shulman responded:  2024-08-23 08:43
You should right-click on the chromatogram graph and choose "Transform > None" in order to see the accurate peak shape.
The chromatogram that Skyline shows you by default is the "interpolated" chromatogram where it has been resampled in the time dimension so that all of the points are evenly spaced. Looking at the interpolated chromatogram gives you a bad indication of the actual peak shape. Usually the interpolated chromatogram is more smooth than the original chromatogram.

Pic4 on the right side looks too rounded as if you might have chosen "Transform > Savitzky-Golay Smoothing".

If you send us your Skyline document and your .raw files we might be able to say more.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
The Share Document dialog also gives you the option to include raw files in the .zip or you could send those raw files to us separately.

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

When you do not have very many points across the peak, the thing that usually suffers is accurate quantification, not necessarily accurate identification.
That is, if you are taking the area under the curve, and the curve is a triangle with only one point above the baseline, you do not really know whether the apex of the triangle is close to the top of the actual elution curve, or was much further down the side.
-- Nick
 
vmohanty responded:  2024-08-23 11:12
Hi Nick,

I have uploaded my files in the URL provided [Pointed)peaks_Aug23_VM]. Please have a look!

Thank you.
Varsha
 
Nick Shulman responded:  2024-08-23 11:57
Thank you for uploading that .sky.zip file with the .raw files.
This does not look like PRM data.

With PRM data, the mass spectrometer has a list of precursor m/z's to isolated, and repeatedly cycles through that list and collects multiple MS2 spectra for each m/z value in that list.
With DDA data, the mass spectrometer looks at each MS1 spectrum, and based on what it sees in that spectrum, decides which m/z's to isolate and fragment.

This looks a lot like DDA data because the exact m/z values of the precursor that was isolated in the MS2 spectra seem to be unique to that particular spectrum. The part that is a little confusing to me is that the m/z values that have been isolated do not match the m/z values of the largest peaks in the MS1 spectra that came before it, so I am not sure how the mass spectrometer is deciding which precursors to fragment.

When you extract MS2 chromatograms from DDA data, the chromatograms always end up looking like that with long straight lines connecting the places where a matching precursor happens to have been isolated.
The chromatograms extracted from DDA data in Skyline are usually not very useful.
-- Nick