Skyline for unknown modifications

Skyline for unknown modifications mprado  2018-10-17

Hi all,

I am wondering if anyone knows if Skyline could be used for the identification of unknown modifications.

For that, I have been running some test to see if I could identify fragment ions without considering the precursor mass, but somehow... I cannot make it work.

Let me explain it with an example. As you can see in the picture attached, I am trying to find several fragment ions of 2 peptides that differs only in one amino acid (the Met at the N-termini): MPLYSVTVK and PLYSVTVK. If in both peptides I only use y1-y7 fragment ions (disregarding the precursor mass), they should be identified at the same retention time. As you can see in the pic attached... This doesn't happen.

For this test I am deactivating the MS1 filtering in Tools>Transition settings>Full-Scan in order to use only the MS2 fragments, but I am not sure if I am missing anything else

Thanks in advance for your help!!

Brendan MacLean responded:  2018-10-18

Hi Miguel,
How are the spectra acquired? Are you using DDA acquisition method?

There is no way to make Skyline extract chromatograms from all MS/MS regardless of precursor m/z. So, the only way you could achieve what you are asking for would be if you acquired your MS/MS in a mode where the mass spectrometer could be expected to fragment the precursor m/z values of 510.2886 and 453.7684 together in the same spectra. That probably limits you to very wide window or all ions DIA, since the two m/z values are 56.5 m/z apart.

What makes you expect your mass spect may have isolated either or both precursors in the same MS/MS spectra?