The MS2_MS2 mode of acquisition for crosslinking analyzes cleavable crosslinkers. It fragments the same precursor ion twice. The first fragmentation (often low-energy CID) cleaves the crosslinker, keeping the two peptides intact. The second fragmentation focuses on fragmentation of the peptides for the peptide database search. An MS2-MS2 experiment using ETD or EThcD for the second fragmentation works best for CID cleavable crosslinks because these crosslinks are stable in ETD and EThcD and result in only two peptide fragment series in the second spectrum.
The following figure gives an example of a crosslink identification the using the MS2_MS2 mode. The spectrum on the top shows the peptides after the cleavage of the crosslinker and the spectrum on the bottom shows the fragment series of both peptides after EThcD fragmentation.
In the case of the MS2_MS2 mode of acquisition for non-cleavable crosslinkers, each type of MS2 is processed separately.