DIA to SRM assay gradient

DIA to SRM assay gradient akhilabrai  2024-01-22 21:37

Hi Skyline team,

I was looking at the tutorial on developing methods for SRM/MRM assay from DIA chromatogram libraries. I had a list of precursors of my interest. It was suggested to run iRT in the qqq instrument. I planned to run Pierce iRT in the Sciex QTRAP in triplicates.
My questions are

  1. What concentration of iRT to be taken?
  2. How to choose the gradient and the run time while running in the QTRAP?

Please help me out.

rj8 responded:  2024-01-23 15:51

The on-column amount of prtc peptides can be any range that gives a good signal. 30-150 fmol on the column should work. I personally inject 3 ul of sample to which I've spiked in 10 fmol/ul of prtc. The gradient is whatever you like. Around here the gradient times will range between 10 minutes and 90 minutes. Whatever gradient you choose should result in at least 6 or 7 data points across the chromatographic peak. Less than that your peak shapes become triangles or quadrangles.

akhilabrai responded:  2024-02-08 03:41

Thank you for your reply.

I had done a trial run of Pierce iRT (200 fmol) in the Sciex QTRAP 6500. We could see the fragments of the peptides in the .wiff file in the analyst. However, when I imported it to Skyline daily, I could not find the transitions. Please help me with that. I am doubting the parameters that I have given in Skyline. I am sharing the skyline document as well as the .wiff file here.

Can you please look into this? Hoping for a positive response.

Nick Shulman responded:  2024-02-08 07:24
I see that this is a DDA experiment where the mass spectrometer decided what to fragment based on what it saw in the MS1 spectra.
In general, Skyline does a bad job extracting MS2 chromatograms from DDA data because a particular precursor does not end up getting sampled at regular intervals. The MS2 chromatograms usually have long straight lines which connect the points where the precursor happened to get selected for fragmentation.

Skyline does allow you to choose "DDA" as the acquisition method at "Settings > Transition Settings > Full Scan", which I see that you have done in your document.
You also need to tell Skyline to extract chromatograms for your precursor. That is, you should right-click on one of the precursors (e.g. "494.0185++ (heavy)") and choose "Pick Children" and check the checkbox next to "precursor".
After you have done that for all of the precursors in your document, you can tell Skyline to extract chromatograms again with the "File > Import > Results" menu item or by going to "Edit > Manage Results" and pushing the "Reimport" button.

It is a bit of a bug that you don't get any MS2 chromatograms when your acquisition method is "DDA" and you do not have any precursors in your document, but, when we first added DDA as an Acquisition Method at "Settings > Transition Settings > Full Scan", we really only intended the DDA chromatogram to be used to help you understand the MS1 chromatogram better.

Skyline is not the best tool for looking at DDA data because most of Skyline's features revolve around chromatograms. Despite this, we do have a few good tutorials for working with DDA data.
The "DDA Search for MS1 Filtering" tutorial will show you how to use Skyline's built-in peptide search engine "MS Amanda":
The "MS1 Full-Scan Filtering" tutorial will show you how to use peptide search results that you might have from a different search engine:

By the way, if you want to send someone your Skyline document you should use the menu item:
File > Share
to create a .zip file containing the Skyline document (.sky file), extracted chromatograms (.skyd file) as well as other supporting files including spectral libraries (.blib).
-- Nick