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.