Figure 2.

Fragmentation spectra of gas-phase cleavable cross-linkers. Upon collision-induced (CID/HCD) or electron-transfer-induced (ETD) dissociation, cleavable cross-linkers break apart, usually at two different positions. This leads to the formation of a characteristic doublet (MS2 level). Alternatively, in the case of protein interaction reporter (PIR) linkers, a reporter ion of a known mass is formed (shown as a red triangle). These ions can be used to unambiguously identify the presence of a cross-linked peptide, which is then selected for further fragmentation to obtain peptide fragments and to identify the amino acid sequence (MS3 level). In the case of stepped HCD or ETD fragmentation, this MS3 level is omitted; instead, the diagnostic cross-linker ions as well as the peptide fragments are detected in the same MS2 spectrum. Peptides are illustrated in green and blue, respectively, the cross-linker is shown in brown, and cleavage sites are shown as red dashed lines.