Tutorial on Prosit use within Skyline? wxxx  2022-03-26
 

Hello,
I would like to be able to submit a list of proteins to Skyline and get an:

  • in silico trypsin digest
  • predicted RT of each tryptic peptide
  • predicted ms2 spectra of each peptide

What I would like to be able to do is to identify 'best' transitions from eac protein (ms1 and ms2) - i.e. those with highest intensity.

Via googling I figured out Prosit can do it. But I cannot find any tutorial which would discuss how to use it within Skyline (either by googling or searching this page). I would need a step-by-step guide - I rarely use Skyline and am not very familiar with it!
Or maybe I should do this by downloading a spectral library?

Could you please point me to any document that mentions prosit?

Thank you.

 
 
pavel shliaha responded:  2022-03-30

I second this request cannot find anything through search on the main site

 
Nick Shulman responded:  2022-03-30
The way to do an in-silico digest in Skyline is to do:
File > Import > FASTA
Skyline will digest the proteins using the enzyme that you have specified at "Settings > Peptide Settings > Digestion", and will apply the filtering criteria specified at "Settings > Peptide Settings > Filter".

If you want to read more about Prosit, here are some things that I found on the Skyline site:
A presentation by Brendan: "Skyline integrates the Prosit prediction server for proteome-wide DIA data analysis using on-demand fragment intensity and iRT prediction":
https://skyline.ms/files/c90e7bac-8854-102e-9e41-e08d31b9c205/%40files/2020-ASMS-Skyline-Prosit.pdf
and a video that goes with it:
https://skyline.ms/files/c90e7bac-8854-102e-9e41-e08d31b9c205/%40files/2020-ASMS-Skyline-Prosit.mp4

2019 ASMS poster by a few people named Tobi:
https://skyline.ms/files/c90e7bac-8854-102e-9e41-e08d31b9c205/%40files/2019-ASBMB-Rohde.pdf

"Using Prosit for PRM assay development and optimization":
https://skyline.ms/files/d1161233-f688-1036-894e-e465a393d561/%40files/Presentations/02-Schmidt.pdf

I do not know very much about how to use Prosit, but if you ask a specific question we can probably figure out the answer.
-- Nick
 
pavel shliaha responded:  2022-03-31
thanks Nick, I actually saw the PROSIT ASMS talk. It was great!

My question is: can PROSIT implementation in skyline predict RT of a peptide based on RTs of peptides in my spectral library, given I have not spiked any standards (like iRTs) into my sample. In other words I have a DDA run of HeLa (without any spikes) can skyline use Prosit to predict RT of a peptide that has not been identified?
 
roman sakson responded:  2022-04-17
Hi Pavel,

I came across this post so I decided to contribute (of course, I am no expert and Nick might correct me if I am wrong). As RT is inherently dependent on your chromatography parameters, I believe that you will always need to provide some benchmark signals, like iRTs, for a prediction tool to work and to provide a RT prediction in minutes. However, if you don't have any spiked standards in your sample, you could provide a set of any peptides from your sample. All you need is a number of peptides (8 - 20) for which you have good IDs and signals, then you could create a "normal" RT-predictor in Skyline from them and provide it to Prosit as a reference. If you have a sample of eukaryotic origin (such as HeLa etc.), Prosit might be able to work with the so-called CiRTs, common iRTs which are peptides shared across eukaryotic species. You can read more about CiRTs here (Parker et al., MCP 2015):

https://www.sciencedirect.com/science/article/pii/S1535947620326335?via%3Dihub

Disclaimer: I have actually never tried this with Prosit myself, with real data in Skyline this would quite certainly work. However, CiRTs are included as a ready-to-go set within Skyline, so you could automatically add them to your document and hope that you will be able to find them in your real data. Then, build a spectral library including your actual peptides of interest, for which you want RT predictions, and choose CiRts (or any other predictor with "iRTs" of your choice) for the library's set of standard peptides.

I hope that this is not too confusing, feel free to ask again!
Roman