I ran a couple of peptides on the LUMOS instrument using the tMSn unscheduled method for collision energy optimization. I used a transition list generated by Skyline with varying collision energies as suggested by the Skyline software.

After analyzing the data in Skyline, I observed the following:

For one peptide, only the precursor ion chromatogram was detected, with very low intensity, and no product ion chromatograms were visible.

For the other peptide, I obtained three precursor ion transitions with intensities around 10⁶, and three product ions, though their intensities were significantly lower (~10³).

I have a couple of questions regarding the method:

Would PRM or a scheduled method be more appropriate for this type of collision energy optimization?

What would be the best approach to optimize collision energy to achieve better product ion intensities?

What can be the reason of not getting the product ions (y ion) on skyline? I do have seen chromatogram in FreeStyle when I put Raw file there.

I am attaching herewith the raw file of the two peptides for your kind perusal.