Hello,

I am working with Hybrid-DIA data acquired on an Orbitrap Exploris mass spectrometer with FAIMS. In principle, we run a DIA-analysis with an inclusion list (n = 50), which targets trigger PRM scans (similar to: https://skyline.ms/_webdav/home/software/Skyline/events/2018%20ASMS%20Course/%40files/Day_2_am_11_15_Jaffe_Hybrid%20PRM-DIA%20Assays.pdf).
The raw files contain standard MS1-overview scans (profile), DIA scans (profile) and target-triggered PRM scans (centroid).
The aim is to compare the number of identified targets from a Hybrid-DIA analysis to a standard native DIA-analysis (without an inclusion list). We use libraries from synthetic heavy labeled peptides of the 50 targets for dotp calculation and identification (threshold: dotp > 0.85).
To get a feeling for the data, we would like to split the Hybrid-DIA raw file into DIA and PRM data to check for peptide targets in both files.
We assume, we identify less targets in the pure DIA data of the Hybrid-DIA file compared to the native DIA-file, as PRM scans should be triggered instead of the DIA acquisition, thereby reducing the scan times for the DIA acquisition. Of course, we expect to detect the targets then in the respective PRM data of the Hybrid-DIA file.

I used Skyline (v23.1) for analyzing the data. As described in the following, I primarily adapted the settings for the acquisition mode (DIA or PRM) and product mass analyzer (Centroided or Orbitrap) to evaluate the data.

(I): DIA-analysis
I loaded the Hybrid-DIA raw file and the native DIA file into a Skyline Template using the setting “DIA” for acquisition mode and then tested two different settings for the product mass analyzer: Centroided or Orbitrap.
For me it was surprising to see transitions using Centroided as the product mass analyzer, especially for the native DIA file, because we record DIA MS2 scans in the profile mode.

Why does Skyline show transitions when using Centroided as the product mass analyzer, although DIA-MS2 was recorded in profile mode?
Does Skyline only show DIA scans or also PRM scans by using “DIA” for acquisition in case of Hybrid-DIA data?

(II): PRM-analysis
I used the same Skyline templates from above and switched from setting “DIA” to “PRM” for acquisition mode (again for either Centroided or Orbitrap) and reloaded the Hybrid-DIA and native DIA raw file.
In both Skyline files (of native DIA + Hybrid-DIA of either Centroided or Orbitrap), I only see the MS1 data and not the MS2. I expected this for the native DIA (because no PRM data), but not for the Hybrid-DIA file.

Why does Skyline not show me the PRM data for the Hybrid-DIA data in this setup?

To rule out the possibility that Skyline is having trouble displaying both DIA and PRM data, I used a Python script with pymsfilereader and MSconvert to filter the .raw files by only keeping the MS1 (n = 2,000 scans) and PRM-MS2 (n = 5,000 scans) scans. The resulting mzML files open fine in ProteoWizard SeeMS — I can clearly see the PRM-MS2 + MS1 spectra (and no DIA-MS2 spectra).

But when importing those mzML into either of the Skyline templates (acquisition mode [DIA or PRM] and product mass analyzer [Centroided or Orbitrap]), hardly any transitions appear. Also, for all Skyline templates I see differences in the number and intensity of transitions – I assume, different filtering is applied.

Overall, my main question is:

Is there a recommended workflow or conversion setting to make Hybrid-DIA PRM scans fully visible in Skyline (either separated in DIA and PRM or combined)?

If required, I can send you the Skyline Templates I used.

Thanks in advance for any help!

Best,
Andreas