About Synapt G2-Si (Waters). Data-independent acquisition

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About Synapt G2-Si (Waters). Data-independent acquisition eriosc  2020-05-05
 

Hello!

I would like to know a few things about Skyline.

I have used commercial software most of the time; however I would like to know if Skyline supports * .raw files from Waters coming from a Synapt G2-Si mass spectrometer. I ask this because I am interested in doing Label-free (DIA) using HDMSE and UDMSE mode. Is it possible to do this?

Additionally, I would like to know if with Skyline it is possible to identify crosslinked peptides (XL-MS) using the same way HDMSE and UDMSE (DIA). If possible, it would be great because I have some ideas to develop using this methodology.

Thanks and regards

 
 
Nick Shulman responded:  2020-05-07
Yes, Skyline can read data from the Synapt G2-Si. I have seen questions posted to this support board from users who were using that instrument.

The next version of Skyline that we are working on right now will have support for crosslinked peptides.
We are still working out the details of how exactly this will interact with results from crosslinking search engines. We are working with some people who have crosslinked peptide search results in something called "proxl" format and I am pretty sure Skyline will be supporting that as a way to be spectral libraries for crosslinked peptides.

Can you tell us a little more about what you would want Skyline to do with crosslinked peptides?
-- Nick
 
eriosc responded:  2020-05-07
The idea of ​​using skyline for crosslinked peptides using DIA in UDMSE mode, was because I read an application note from Waters Corporation called: "acquisition mode characterization for the qunatitative and qualitative analysis of cross-linked peptides by target and untarget LC- IM-MS. " and that it uses Skyline for the assignment of the ionic series; although I think they used ETD fragmentation. I know that to perform XL-MS, the workflow is done in DDA mode, but since I work in DIA mode (I feel that it is very robust to perform Label-Free) I thought that maybe the current versions of Skyline already support it and I was excited because they combined the way of working that I usually work in the lab with something that I want to exploit. My idea is to clone 2 proteins of my interest to interact and cross-link them with Disuccinimidyl suberate (DSS), enzymatically generate the cross-linked peptides to be analyzed. Using Ion mobility I can generate charge dependent clusters, and extract the mobility information of the ions of interest, in this case use peptides with charge state z = 3 + or higher; Then I can create a method in UDMSE mode to apply quasi-specific collision energy to crosslinked peptides and gain selectivity. What I would like skyline to do is to deconvolve the ions to be able to assign the ion series of the cross-linked peptides, in a other words, to know which peptides of the "X" and "Y" protein are cross-linked.

How long will it take to release the version you are telling me about?

Cheers!
 
Nick Shulman responded:  2020-05-07
On that poster, it looks like they have told Skyline that these crosslinked peptides are "small molecules", where they have told Skyline the chemical formula of the intact molecule, and all of the fragments to look for. That certainly works today. It looks like the poster might be saying that they have some python scripts that they wrote to help with calculating the fragment ions.

We might release a Skyline-Daily with some crosslinking support in about a month. Skyline 20.2 will be the next official release of Skyline, which will probably happen late in the summer.
-- Nick