DDA --> PRM not working sa825  2019-09-19
 

Good afternoon,

I am trying to conduct PRM on a Thermo Q Exacitive for a list of proteins/peptides. They are heavy labelled so I am really interested in picking up the heavy peptides.

To do this, I first conducted DDA using the sample of interest on the Thermo and detected the many of the proteins/peptides of interest (Skyline shows that their precursors M, M+1 and M+2 are present)

However, when I run a Standard PRM (Exported a single method, standard isolation list) on the same instrument, using the same sample, only ~2 product ions are detected.

Can you please explain to me why this is the case? Maybe its a setting I have missed out?

I have attached a zip file containing the situation on the File sharing Page and titled it: "DDA -> PRM Not working"

Thanks and I look forward to your reply,

Shimon

 
 
Nick Shulman responded:  2019-09-19
Shimon,

Can you send us that file "SA_190919_MP_Proteins_Heavy_PRM.raw"?

I see that nearly all of your MS2 chromatograms are completely flat. One thing that could cause this would be the mass accuracy that you specified at:
Settings > Transition Settings > Full Scan > MS/MS Filtering Mass Accuracy

You have that set to 10ppm. If your mass spectrometer was not actually not that accurate, maybe because it was not properly calibrated, then that could result in all of your chromatograms being flat.

I think it will be easier to figure out what is going wrong if we see that .raw file.
-- Nick
 
sa825 responded:  2019-09-19
Hi Nick,

Thanks for your reply.
I have uploaded the PRM file to the file sharing page as a zipped folder.
The description is: "SA PRM Raw File"

Thanks,

Shimon
 
Nick Shulman responded:  2019-09-19
Shimon,

Are your DDA results in the .sky.zip that you sent us? Were they "JODIE_SA_180819_MP_HEAVY" and "JODIE_SA_180819_MP_HEAVYB"?

How did you identify the peptides in your DDA sample? Did you use a peptide search engine such as Sequest? Or did you just look for coeluting heavy and light MS1 chromatograms?

The simplest reason for why you are not seeing MS2 signal in your PRM experiment might be that those particular peptides are not present.

If you want you could send us your DDA peptide search results and/or your DDA raw files.
-- Nick
 
sa825 responded:  2019-09-20
Hi Nick,

Yes they are in the zip file. I have tried to attach the DDA raw files on the File Sharing page but the page keeps on crashing (I think the files are too large) Is there another way to get the files to you?

No I didn't use a peptide search engine. I looked for co-eluting heavy precursors. Cause the sample is heavy labelled such that there is close to 100% labelling, I'm not expecting to see a lot of light peaks. In case where light peaks were detected, I did also look for co-eluting heavy and light MS1 chromatograms.

And I see, if the peptides are not present, why are they being detected in MS1 on Skyline?

Thanks,

Shimon
 
Nick Shulman responded:  2019-09-20
It might be easier for you to send files using Google Drive or DropBox. I'll send you an email.

I am not sure how likely it would be to see two things in MS1 that looked like coeluting heavy/light pairs but were misidentified. Did you spike in specific heavy peptides, or did you do something like label a whole organism.
-- Nick
 
sa825 responded:  2019-09-20
Alright please do.

I’m not sure I did any of your two options. I think it’s more appropriate to say I heavy labelled a sample after tryptic digestion. I hope that makes sense?

Also, I have just sent you the files using something called ZendTo. Let me know if you have been able to pick them up.

Thanks,

Shimon
 
Brendan MacLean responded:  2019-09-20
I would still recommend running a peptide search for the heavy labeled peptides in your DDA data. Skyline will perform much better on DDA data if you have peptide spectrum matches. Trying to match only heavy and light precursor isotopes, especially when you may not be seeing the light form is asking for problems. We determined this quite clearly during the 2013 ABRF sPRG study:

https://panoramaweb.org/project/ABRF%20sPRG/begin.view?

Which unfortunately never got published. In this case, participants injected a known standard mix of 1,000 heavy labeled peptides (so expected to be detectable) into a HeLa background to try to quantify the 1,000 matching unlabeled analyte peptides.

Many people were reasonably successful just searching their DDA MS/MS spectra with a Human FASTA file. but one of the most successful labs searched only for the heavy labeled forms of the peptides, got IDs for most of them and then used that information in an Import > Peptide Search in Skyline with retention times determined by the DDA spectrum matches for the heavy labeled standards.

I have attached a slide that I still present of the study results and also my main take-aways for how to achieve success with Skyline on DDA data.

You might also watch the first Skyline Tutorial Webinar on DDA data, which covers similar topics:

https://skyline.ms/webinar01.url

Hope this helps. I don't think it is a good idea to continue troubleshooting your use of Skyline on DDA data without peptide spectrum matching, and without even labeled standard peptides, but instead just a sample mixture where one sample is labeled during sample handling.

Thanks for posting to the Skyline support board.

--Brendan
 
Nick Shulman responded:  2019-09-20
Shimon,

Thank you for sending me your .raw files.

I believe that you have misidentified the peptides and that is why you are unable to find any MS2 signal for the ions that you are monitoring.
-- Nick
 
Brendan MacLean responded:  2019-09-21
One more thing to be aware of, which has confused others in the past. Skyline uses 0.5 m/z as the default MS/MS peak matching tolerance, which is totally inappropriate for high resolution, high accuracy mass analyzers like the Orbitrap and modern TOF instruments. So, if your MS/MS were collected in a modern high-resolution mass analyzer, then I usually recommend reducing the "Ion match tolerance" on the Transition Settings - Library tab to at least 0.05, which is roughly 50 ppm. Unfortunately, this is a constant, while PPM varies with the m/z. I have been thinking of making it possible to switch this tolerance number to PPM, since that will make it match with the chromatogram extraction "Mass Accuracy" number on the Transition Settings - Full-Scan tab when mass analyzer is set to "Centroided".

The problem with leaving the tolerance at 0.5 m/z moving between DDA and PRM is that you can end up matching ions to peaks that are as far away as 500 PPM from where you will extract chromatograms from PRM spectra. So, Skyline will pick to target fragment ions which were never really seen in your DDA data, just some intense peak up to 500 PPM from the ion chosen.

So, you may have misidentified MS/MS spectra or in some cases, you may just have misinformed Skyline about the accuracy of your DDA MS/MS.

Hope all our feedback somehow gets you on track to correctly identify and extract chromatograms for your targets of interest. Thanks for posting to the Skyline support board and supplying your example files.

--Brendan