| Nick Shulman responded: |
2018-07-30 11:52 |
I do not understand your question, but it might help if you could send us your Skyline file.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files, including spectral libraries.
You can upload that .zip file here:
https://skyline.ms/files.url
Let us know which peptides we should look at. |
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| dengjingjing01 responded: |
2018-08-01 12:48 |
Hi Nick,
Thanks for your reply. I have uploaded the .zip file. The file name is 08012018_public library.sky.
Here I am also attaching a .ppt file to explain more. The first slide shows the two peptides I am interested. The second and the third slides show the spectrum data in GPM website. Although I include all the mouse spectrum libraries from GPM in skyline, it shows spectrum information unavailable.
Please let me know if you need any other information. Thanks.
Best,
Jingjing |
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| Nick Shulman responded: |
2018-08-01 13:35 |
Yes, the peptide "ASSMDEQQQTEFYDAVK" is not in any of the spectral libraries that you have.
You can see which peptides are in the library by going to:
View > Spectral Libraries
also, the Nist libraries (*.msp) are just text files, so you can open them using a text editor such as Notepad.exe and see that the peptide is not in there.
I do not know what the relationship is the between the GPM website and the NIST (*.msp) and X!Hunter (*.hlf) libraries that you have, so I do not know why the libraries do not contain that peptide. |
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| Brendan MacLean responded: |
2018-08-01 14:27 |
Hi Jingjing,
Your document is referencing 28 different libraries. This is likely not a good strategy for targeted proteomics. You will be far better off if you use libraries you know positively mimic the methods you hope to apply in your own mass spectrometry. Your approach to libraries seems to be much more one of trying everything that is possible without much reference to what you will do yourself in your experiment. For instance,
...
hcd_itraq_selected
hcd_itraq_phospho
hcd_selected
consensus_final_true
...
Are you expecting to use iTRAQ labeling or phospho enrichment? You are using 1.2 GB of .msp library files from iTRAQ studies.
Okay, I see that the .hlf files are for each separate chromosome of a mouse (1-19, X, Y, MT, other). I wonder if there is a single library you could be using from theGPM. As from Nick, I am not sure the expected relationship between the online database and the library files. It seems like you might figure out which chromosome you expect this protein to come from and then use View > Spectral Libraries to see if you can find the peptide in that specific library. If you can't, as Nick suggests, then you can contact theGPM to ask why.
Thanks for the files and PowerPoint slides.
--Brendan |
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| dengjingjing01 responded: |
2018-08-02 08:56 |
Hi Nick and Brendan,
Thanks for both of your replies. The .hlf files contain all the mouse database I can find from GPM. I am interested in multiple proteins. Sorry I didn't include all the protein names here because we are asked to keep the proteins confidential requested from our collaborator. The proteins come from different chromosomes, and that is why I am trying to include all the database I can find. I will ask GPM what is the relationship between the online database and the library files.
BTW: I am doing label free without enrichment. I should not include the iTRAQ labeling or the phospho database.
For further steps, may I ask you some advice?
1) Is DDA method necessary for me to create my own library? I don't think I can coverage all the proteins I am interested in since some protein is quite low aboundent.
2) If neither my own DDA method nor the public library has the spectrum for what I am interested in, is the only way synthetic peptide to solve this problem?
3) Although skyline can give theoretical fragmentation information, I think it is risky for me to identify or quantify the peptide I am interested in just based on the match of my real data to theoretical information, but not to library data. Am I right?
Best,
Jingjing |
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| Brendan MacLean responded: |
2018-08-05 12:30 |
Hi Jingjing,
Some responses to your most recent questions:
- Skyline makes it possible to generate libraries from targeted runs processed with Skyline in two ways: a. File > Export > Spectral Library or b. building a "Chromatogram Library" on Panorama (https://panoramaweb.org/tutorial_clib.url). These do not require DDA. You could also produce spectral libraries from PRM data or DIA data reduced to deconvoluted spectra by a tool like DIA-Umpire.
- Synthetic peptides or expressed proteins are a good way to get high confidence spectra for your peptides of interest. If you have any other way to purify a sample for your protein of interest to increase your confidence in what you are measuring, you might also be able to just target your peptides of interest and then export a library as described in #1. You will just need to justify why you are confident the measurements represent your peptides.
- Skyline does not predict theoretical ion abundances. I agree that it is risky to base conclusions on peak measurements when you have none of the following: a. a stable isotope labeled reference peptide, b. empirically measured relative fragment ion abundance, c. empirically measured relative retention time.
Hope this helps. Good luck with your research.
--Brendan
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| dengjingjing01 responded: |
2018-08-08 09:04 |
Hi Brendan,
Thanks a lot for your answer. All of them are quite useful.
Enjoy your day!
Best,
Jingjing
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08012018_public library.pptx