Targetted MS Analysis Using MS1/MS2 Ion's Presence/Absencse

Targetted MS Analysis Using MS1/MS2 Ion's Presence/Absencse csb548  2023-07-10 03:32

Hi everyone,

I'm a PhD student trying to develop a fast, high-throughput analysis of a particular enamel protein. I'm currently analysing my tims-TOF MS data with PEAKS 11 which works great but is a bit slow for a method that is supposed to be fast. I'm very new to skyline so not sure of what the program is capable of so thought I'd come here to ask some questions!

I am looking for software that allows me to basically input an MS1/MS2 m/z that I'm expecting to see (dependent on sample) and tells me if that m/z is present or not. Is skyline able to do this?


csb548 responded:  2023-07-10 03:35

Just for a bit of extra context if it is helpful. My project is looking at sex determination based on the proteins found (as some are X-chromosome linked, and some are Y-chromosome linked). Since biological females won't possess an Y-chromosome, they don't have the Y-isoform of the protein. Therefore, a rapid analysis that could tell me if the Y-isoform was present in the MS data or not (by m/z values) would be super helpful (I'm just not sure if it is possible)!


Brian Pratt responded:  2023-07-10 14:24

Yes! Skyline has been used for this very thing:

Thanks for using the Skyline support board,

Brian Pratt

csb548 responded:  2023-07-11 01:48

Thanks for your response! I saw this paper just before I posted my question as it was what gave me the idea of looking more into Skyline. I'm not sure if they've used Skyline in the way I was interesting in though as they weren't focused on fast analysis. I was trying to figure out if it is possible to essentially screen a library of MS and look for a few set MS1/MS2 m/z values in all of the spectra and get basically a 'yes or no' as to whether those m/z values are in each of the sample's MS/MS (and therefore if the corresponding sex peptides are also present in each sample).

I'm not sure if that fully makes sense or if it's possible so that's why I'm here!


Brian Pratt responded:  2023-07-11 07:21

Yes, Skyline is built for exactly that kind of targeted analysis, and its rich scripting and reporting capabilities make it ideal for this kind of pipeline integration.

Mike MacCoss responded:  2023-07-11 10:20

We've done this a couple of times in the past. The best way to do this is to make a spectral library with the Y-chromosome specific proteins/peptides -- we've used Prosit for this. You can then calibrate the iRT to know at what RT those peptides should appear. If the data are collected using DIA you can use the mProphet scoring or the EncyclopeDIA search tool that is part of Skyline.

If your data was collected with DDA then you can make a fasta with just these Y-chromosome proteins and you can search those using the internal search tools.

We definitely recommend using Skyline to do the mass calculations.

Hope this helps,