Dear Skyline support team and Brendan,

I had a course in targeted proteomics in Zurich last jun. Now I am trying to apply it to my experiment. I am working with phosphorylated proteomics. I have 4 biological replicates (each replicate with 2 fractions) comparing Jurkat T-cells conntrol vs cisplatin treated. After LC-MS/MS run, I performed search on Mascot using the default (0.05 Da and 10 ppm) against human database. After that I loaded the searched files on Skyline (DIA data with DDA spectral library) with the raw files to extract chromatogram.

Peptide setting was set up with parameters as the tutorial for *PRM refinement*. Transition setting was set up with 0.01 m/z for ion match tolerance instead of 0.05. I supposed this would be better for me to filter the data. For instrument in transition setting I used 0.055 m/z for method match tolerance as default.
Full scan setting as in the attachment. I followed mostly all of the parts in tutorials Targeted MS/MS and PRM refinement. However, I started with threshold for dopt and idotp values as 0.5 and 0.3. I want to have a big list of peptides before I can narrow it down in the next run.

So, I performed the first PRM run using the isolation list exported from Skyline with respective RT for each peptide and RT window as 8 mins. The first PRM run was performed using Jurkat control T-cells without treatment and enrichment. As I supposed that PRM can help me detect these phosphorylated peptides and their non-phosphorylated peptides without the need of enrichment (Purpose of using targeted proteomics, right?). AS the DIA data was obtained from 4 hours run in Qex. So, I used the same 4 hours gradient for the first PRM.

After the run, I did the same thing as before and loaded the searched file on Skyline using (PRM was choose in the build spectral library window). I did everything as the same like before and I got the list of 8 peptides and 8 proteins. Only 2 out of 8 are phosphorylated peptides. I understand that phosphorylated peptides will not be ionized efficiently where there are much more abundance non-phosporylated peptides. But with the scope of my experiment. I have to run them with both phosphorylated and non-phosphorylated peptides to compare.

So my question is: Do I have to set up some different setting when I import PRM data on skyline rather than using the same setting for DIA? Because I checked the mascot run from PRM run, and I saw more phosphorylated peptides on that list. When I built spectral library for PRM data. I got a message (Spectra matching the following peptides had multiple ambigous peptide matches and were excluded, also can be seen in the attachment). Now, this could potentially be a big problem for me as I am losing my peptides here. Do you know how to fix this problem? Also in the DIA data set, I saw both the precursor peaks and transition peaks,because these are DIA data, so, I understand that. However, the peak intensities for precursor ions are way more abundance than the peak intensities for transition ions. So, I don't know if this is a normal thing or if the fragmentation was not good enough to break these precursor ions down ?

You can find the supported data in the attachment. I really hope that you can help me with these problems. Thanks so much. I really appreciate your help.

Best,

Trung