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Inquiry on Bottom-Up Analysis Using Skyline for Protein/Peptide Identification

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Inquiry on Bottom-Up Analysis Using Skyline for Protein/Peptide Identification wei ng2  2025-03-06 18:18
 

Dear Support Team,

I hope this message finds you well. I have recently obtained results from LC-QTOF analysis and am seeking to determine the potential proteins and peptides present in my sample. I would like to inquire whether it is possible to perform a bottom-up analysis using Skyline for the identification of these proteins and peptides.

If this is feasible, I would greatly appreciate it if you could recommend any relevant tutorials or instructional videos to guide me through the process.

Thank you for your assistance, and I look forward to your response.

Best regards,
Smith 13
 
 
Nick Shulman responded:  2025-03-06 18:55
It sounds like you have probably run a DDA experiment where the mass spectrometer was told to fragment the most intense MS1 fragments for the purpose of identification.

The DDA Search tutorial will show you how to run a peptide search using "MS Amanda" which is included with Skyline:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_dda_search

Let us know if you get stuck. If you want to send us your files you can use the "File > Share" menu item to create a .zip file containing your Skyline document and supporting files including spectral libraries and extracted chromatograms.

Files which are less than 50MB can be attached to these support requests.
You can always upload larger files here:
https://skyline.ms/files.url
-- Nick
 
wei ng2 responded:  2025-07-03 00:18
Dear Nick,

Thank you very much for your guidance. I have followed the instructions provided in the tutorial and was able to successfully replicate the results shown. I truly appreciate your support.

I do, however, have a few questions that I hope you could help me clarify:

1. Based on the screenshot I have attached, does the list of proteins/peptides shown on the left-hand side represent all the proteins/peptides that have been identified as present in the sample?

2. Is it possible to determine the confidence level and the quantity or relative abundance of these proteins/peptides using Skyline? If so, could you kindly advise where I might find this information within the software?

3. Lastly, could you recommend a suitable protein database for working with microalgae samples?

Thank you again for your time and assistance, and I apologize for any inconvenience caused.

Kind regards,
Smith 13