some peptides show incomplete peaks

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some peptides show incomplete peaks lvyayao90  2019-04-23
 

Hi Skyline team
When I imported my results into the Skyline, some peptides show incomplete peaks, I pasted one in the word file below. Can you please explain it for me and is there anything I can do to avoid this?
Thank you very much!

Yayao

 
 
Nick Shulman responded:  2019-04-23
Can you send us your whole Skyline document?

In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that file is less than 50MB you can attach it to this support request. Otherwise, you can upload that file here:
https://skyline.ms/files.url

I cannot tell for sure what is going on from the picture that you attached. Here are some things that could be going on:
1. You might be trying to extract MS2 chromatograms from DDA data. Because DDA does not sample precursors on any sort of predictable schedule, MS2 chromatograms extracted from DDA data may appear to be truncated, and may have long stretches of retention time without any points. You should tell Skyline to only extract MS1 chromatograms from DDA data.
2. You might have set the Retention Time Filtering to a really small number. That setting is at "Settings > Transition Settings > Full Scan".

I'll know for sure what's going on when I see your Skyline document.
-- Nick
 
lvyayao90 responded:  2019-04-23
Hi Nick
I have uploaded the Skyline document, the PRM list and a word file including extracted chromatograms in the .zip file as you asked.
About the two things you mentioned above, the file i imported was a PRM file and the Retention time filtering was 5 (i tried 50, nothing changed).
And i am using Skyline-daily(64-bit) 4.2.1.19095

Yayao
 
Nick Shulman responded:  2019-04-24
Thanks for sending that .zip file.
Can you send me the files "6600_QconCAT_PRM_20190411_1000.wiff" and "6600_QconCAT_PRM_20190411_1000.wiff.scan"?

I think what is happening is that your PRM method only told the mass spectrometer to collect data for any particular precursor over a very short retention time range. You might see longer chromatogram traces in other programs, because other programs have a different way of deciding which MS2 scans belong to the precursor that you have asked for a chromatogram for.

By the way, I noticed that your Transition Full Scan settings are telling Skyline to extract MS1 chromatogams, but you do not have any precursor transitions in your document.
If you go to:
Settings > Transition Settings > Filter
and change "Ion Types" to something like "y, b, p" (you currently have it set to "y, b"), then Skyline will show you MS1 chromatograms.

Also, for a scheduled PRM method like you have, you should probably set the "Retention Time Filtering" on "Settings > Transition Settings > Full Scan" to "Include all matching scans" (even though that setting turns red when you choose it). This setting probably has nothing to do with the behavior you are seeing, but that's what the setting should be if you have already told the mass spectrometer the time range over which to collect PRM scans.
-- Nick
 
lvyayao90 responded:  2019-04-25
Thank you very much for correcting the details for me!

The two files are uploaded in a file named 6600_QconCAT_PRM_1000.rar

Also, you can see I set the retention time range as 6 minutes in the image attached below(I am using Triple TOF 6600 of AB sciex), it's for every precursor. I don't quite understand why Skyline shows only 20 seconds of the chromatogrames like peptide STWLILHHK and shows 20 minutes of the chromatogrames like peptide IGSTPVLVLSR, if it doesn't bother too much, would you explain that for me? thanks a lot!

Yayao
 
Nick Shulman responded:  2019-04-25

Yayao,

I do not know very much about setting up a mass spectrometer method, but I do not think the "Include List" is the right thing for a PRM method.

Your include list has mass 862.879 at time 30.090 minutes.
I can see that your .wiff file has several MS2 scans with a precursors and times that are very close to that:

TimePrecursor m/z
30.309~862.87637
30.34933333~862.87923
30.38965~862.87821
30.42998333~862.87838
30.47031667~862.87901
30.51048333~862.87722
30.55065~862.87917
30.59081667~862.88255
30.63096667~862.87978
30.67613333~862.87892

Normally, with a PRM method, exactly the same precursor value (862.879) would get selected for fragmentation over the course of several minutes. Instead, it looks like your mass spectrometer was selecting whatever m/z was detected near to your specified value.

I hope you can figure out how to set up your method for PRM.
-- Nick

 
Brendan MacLean responded:  2019-04-25

SCIEX calls it MRM-HR. This might be a good place to start on how to set up a scheduled MRM-HR method:

https://sciex.com/x32887