Hello,

I generated a peptide mapping dataset using my Agilent 6545 QTOF. The method is an "all-ions" (MSe / MS All) type of experiment, where the 1st scan is a low energy 0V CE, and the next three scans are 15,30,and 45V high energy scans.

I am having issues with:

1. Searching the data
2. Viewing the different high energy scans.

I have been able to make some progress with #2. I used the "Edit Spectrum Filter" option to have Skyline give me peptides for each collision energy. However when select the MS/MS spectrum for my peptides, I always see that the Aquisition information shows the Collision Energy at 15V. This is my first high energy scan. I have to believe that I would get optimal fragmentation information from my 30 and 45V scans as my peptides increase in size. Ideally Skyline should be picking the CE with the most intense fragment ions, but I don't believe that is happening.

For 1, I have no idea where to begin. I thought perhaps Skyline might be able to perform an all-ions based search as it is similar to waters MSe data. But it keeps asking me for an isolation scheme when I chose the type of mass spectrometer. I am unsure how to proceed here. I am also engaging Agilent on this as well (they aren't even sure that I can perform a peptide search on this data at the moment)

You might ask why do this? Well the cycle time is decently fast, like 0.5s per cycle. So we get 10-12 points across our 6s wide UPLC peptide peaks, which is nice. I don't think Agilent's AutoMS/MS can move that fast.

Anyways I digress. Just looking for some help here