Identifying proteins

support
Identifying proteins p pereira79  2020-07-14
 

Dear ones,

I am using Skyline to identify proteins from a TOF analysis.
Well, I'm using a UniProt FASTA file as a library (75,000 proteins, and countless peptides), and the upload of RAW files takes too long time (it's taking more than 24 h just one file of size 1.32 Gb!).
Then, I would like to know:

  • Does Skyline work without internet?
  • Why is the upload taking so long?
  • Is it possible for the skyline to connect with a library (like Uniprot) online?

Best regard.

 
 
Brendan MacLean responded:  2020-07-14

Hi Paulo,
You are going to need to do some of the tutorials and learn a bit more about Skyline. Your document has 2.2 million peptides and 13 million transitions.

Depending on what you are actually trying to achieve, Skyline may or may not be a good choice of tools for you. Generally for proteomewide work, you want to first build a spectral library out of DDA data. If you are analyzing the DDA data through chromatogram extraction from the MS1 spectra, then you can just continue from there with only the peptides detected in the DDA data. If you are analyzing DIA data, then you can use the DDA library to query the DIA MS/MS spectra.

So, is this a DDA experiment? In which case you might start with these resources:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ms1_filtering
https://skyline.ms/webinar01.url
https://skyline.ms/webinar08.url

Or a DIA experiment? in which case you might start with these resources:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_dia_swath
https://skyline.ms/webinar18.url
https://skyline.ms/webinar14.url
https://skyline.ms/webinar15.url

Hope these help get you going in a more productive direction with Skyline. Thanks for posting to the support board.

--Brendan