Product Ion Selection Without Spectral Library

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Product Ion Selection Without Spectral Library sa825  2019-08-29
 

Good afternoon,

I wanted to ask how I can select the best product ion for a peptide in Skyline?

I have been trying to detect heavy labelled peptides using PRM on a Thermo Q Exacitive. These peptides have already been detected using DDA on the same instrument but when I run PRM, I don't get detection of all the peptides and they are sometimes detected at different retention times to what is expected.

To overcome this issue, I considered that there were possibly too many precursors being viewed within the same time window on the Thermo so I have selected 1 precursor per peptide per protein. However, this has only yielded a slight improvement in detection.

I am now considering reducing the number of product ions per precursor to just 1 per precursor. What do you think of this and if so how do I select the best product ion? I have done the Method Editing Tutorial so I know I can rank the product ions using a spectral library but I didn't use one when I created my peptide list as I was trying to do a targeted approach so using one here doesn't seem the right option?

I really hope all of that makes sense?

Thanks,

Shimon

 
 
Brendan MacLean responded:  2019-08-29

Hi Shimon,
If you are doing PRM it makes no sense at all to limit your transitions, because only the time spent on each precursor matters. Once you have acquired MS/MS spectra for your precursor of interest it doesn't matter how many fragment ions you extract from it. This is all done in software and has no impact on the acquisition.

You can run your PRM data through the same peptide spectrum matching software you have used on your DDA data, and it should match spectra in your PRM data to your peptides of interest.

If you feel that the quality of extracted chromatograms is not good, then there may be a problem in your Skyline settings.

Can you provide your Skyline document (using File > Share - Complete to create a .sky.zip file) which you can post directly with your next response or if the files is larger than 50 MB, you can upload it to:

https://skyline.ms/files.url

Maybe point to some specific peptides you feel are problematic. Or you could also provide screenshots of your chromatograms and Transition Settings - Full-Scan tab. You haven't really given us that much to go on.

Thanks for posting to the Skyline support board.

--Brendan

 
sa825 responded:  2019-08-30

Hi Brendan,

I see what you're saying.

I have attached my Skyline Document (Best Peptides Monoisotopic) to the File Sharing Folder for you o have a look at the Skyline Settings in relation o the issues I'm experiencing.
The first tab in Skyline is the DDA file and the 2nd is the PRM file.

Thanks and I look forward to your response.

Shimon

 
sa825 responded:  2019-09-06

Hi Brendan,

After further discussion in my lab, I have realised that the first tab is actually an Uncheduled PRM and the 2nd is a Scheduled PRM.
The issue is that I'm seeing the peptides of interest in the Unscheduled PRM but not in the Scheduled PRM?

Thanks,

Shimon

 
Nick Shulman responded:  2019-09-09
Shimon,

Thank you for sending us your Skyline document.

My guess is that the reason that you are not seeing your peptides in your scheduled PRM run is that your PRM method did not tell the mass spectrometer to collect MS2 scans over the retention times when your peptide was actually eluting.

How did you generate your PRM method?

I see that your Skyline document is using the SSRCalc retention time predictor. My understanding is that the retention times predicted by the SSRCalc in Skyline are not very accurate and are not suitable for scheduling a PRM method.

By the way, I noticed that at:
Settings > Transition Settings > Full Scan
you have "MS1 filtering isotope peaks included" set to "None".

If MS1 scans were collected in your PRM method, you should probably change that setting to something else. When that is set to "None", the precursor transitions in your Targets tree end up measuring the intact precursor from MS2 scans. If you change that to something else, then you will get chromatograms extracted from the MS1 scans.

I hope this helps. We might be able to give you better answers if we see your .raw files. You can upload them here:
https://skyline.ms/files.url
-- Nick