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Problems (PRM workflow and isotope tags targeting cysteine residue)

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Problems (PRM workflow and isotope tags targeting cysteine residue) ylam  2018-04-24 14:20
 
Hello:
 
I have a question regarding Skyline and am wondering if you could help me out.
 
I used Skyline a couple of years ago, and it interpreted the data very well for one of our studies. Thanks for your help at that time!
 
1) I am now trying to use Skyline again for another study, and I am having several problems/questions.
I am using the PRM workflow and trying to import the Proteome Discoverer 1.4 msf file into the Skyline. When I input the sequence of the protein of interest with trypsin as an option (Import FASTA), it says the protein only has 4 peptides (see attached: SkylineQuestion.jpg). However, the protein clearly has more than 8 peptides, how come? The sequence is listed below:
 
>sp|P12931.3|SRC_HUMAN RecName: Full=Proto-oncogene tyrosine-protein kinase Src; AltName: Full=Proto-oncogene c-Src; AltName: Full=pp60c-src; Short=p60-Src
MGSNKSKPKDASQRRRSLEPAENVHGAGGGAFPASQTPSKPASADGHRGPSAAFAPAAAEPKLFGGFNSS
DTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLSTGQTGYIPSNYV
APSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKL
DSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWEIPRESLRLEVKLGQGC
FGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMSKGSLL
DFLKGETGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEYT
ARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPEC
PESLHDLMCQCWRKEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL
 

2) It looks like the Skyline was not able to incorporate all the peptide identification from Proteome Discoverer results, although I have set the cut-off score = 0, when I imported the results.

3) We are using a cysteine-reactive tag C(8)H(10)O(2) [138.06808 Da] and its isotope (deuterium) counterpart 2H(6)C(8)H(4)O(2) [144.105740 Da] for our experiments. I have set these two as variable modification and 2H(6)C(8)H(4)O(2) - C(8)H(10)O(2) = 6.03766 Da as "heavy isotope modification". But Skyline seems to also see 144.105740 Da + 6.03766 Da as "heavy" as well.



It has been a while since I used the Skyline, any help will be greatly appreciated!
 
Best wishes,
Wai
Ying Wai Lam
VGN Proteomics Facility
University of Vermont
802-656-4709
 
 
Nick Shulman responded:  2018-04-24 14:47
I get 23 peptides when I insert that FASTA record into a Skyline document. My guess is that it something about your settings at:
Settings > Peptide Settings > Filter
which is causing Skyline to not give you all of the peptides that you want.

It is also possible that you have "Pick Peptides Matching Library" turned on at:
Settings > Peptide Settings > Library
In that case, Skyline will only give you peptides which Skyline finds in your spectral library.

I do not understand your other questions. It would help if you could send us your 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 .zip file is less than 50MB, you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

Also, you should send us your ProteomeDiscoverer files and we will figure out why you are not seeing what you expect to see.
 
ylam responded:  2018-04-24 15:27
Hello Nick,

Thanks for your quick reply!

Attached are:
1) the Skyline document and supporting files
2) Proteome Discoverer 1.4 msf file,
3) All the m/z values that were used for the targeted analysis on Q-Exactive

We use two cysteine sulfenic acid -reactive tags for quantitative analysis, one is dimedone (MW: 138.068) and the other one is dimedone-d6 (MW: 144.105740). The pair should have a 6.03766 Da difference.

We basically identified all the dimedone or dimedone-d6 containing peptides in our protein by SEQUEST with Proteome Discoverer, but somehow the results cannot be incorporated into the Skyline. I have set the cut-off value to zero.

I did similar workflow with SILAC-labeled phosphopeptides (incorporating Proteome Discoverer 1.4 results into Skyline) a couple of years ago, it worked pretty smoothly.

Many Thanks!
Wai
 
ylam responded:  2018-04-24 15:36
Hello Nick,

It gave me an error when I was trying to attach the files to my previous message. I have uploaded all the files instead using the link you provided: https://skyline.ms/files.url.

Thanks very much for your help!!

Wai
 
Nick Shulman responded:  2018-04-24 16:08
Thank you for sending those files.

One thing that you need to do is uncheck the checkbox next to the "Carbomidomethyl (C)" modification in:
Settings > Peptide Settings > Modifications

"Carbomidomethyl (C)" is a "static" modification, which means that Skyline thinks it should be applied to every Cysteine in your document. In some experiments, that is a good thing, but in your experiment, you chemically treated the peptides differently, and you do not want that modification applied to everything.

Also, you should probably get rid of the "dimedone-d6-whole" modification from your "Structural Modifications". When you defined that modification, you did not check the "variable" box, which makes Skyline think that, just like "Carbomidomethyl (C)" it needs to be applied to every cysteine.
 
Brendan MacLean responded:  2018-04-24 16:50
You may also find the Skyline Tutorial Webinar #10 "Working with Modifications in Skyline" helpful in understanding what is possible in Skyline with modifications.

http://skyline.ms/webinar10.url