UVPD fragmentation of cross-linked peptides with thiosuccinimide and thiazine linkers
Ultraviolet photodissociation (UVPD) has been applied for the identification of disulfide heterogeneity and non-native crosslinks such as trisulfide and thioether bonds which are generated during the production of and storage of monoclonal antibodies 21. UVPD at 213 nm can be used to selectively dissociate these bonds to produce characteristic fragmentation patterns which are derived from the homolytic cleavage of S-S and C-S linkages 22. Based on these previous observations, we examined the issue of whether or not conjugates containing thiosuccinimide or thiazine linkers could be distinguished by the UVPD method incorporated in LC/ESI-MS/MS (see Experimental section). For P2-P4, product ions at m/z 813.42, 847.41 and 1260.60 in Figure S16a, observed for the thiosuccinimide form, were similar to those observed in MALDI-MS/MS (Figure S14), which most likely had arisen from a 1, 4-elimination (Figure 4). Note that the observed product ions were not those derived from homolytic cleavage at the C-S linkage of the thiosuccinimide, since they were smaller by 1 Da than those from the homolytic cleavage. These ions were sufficient for identifying the thiosuccinimide form, whereas, UVPD-MS/MS of the thiazine form did not give any product ions derived from the cleavage at the pseudopeptide bond which was newly formed in the thiazine form (Figure S16b). Considering the conditions of the UVPD used in this study, i.e., photoactivation time (500 ms) and the method of ion trapping in the device, it is likely that the precursor ions primarily underwent spontaneous fragmentation by 1, 4- elimination rather than dissociation by UV-laser irradiation.