Conclusions:
MALDI-MS analysis can be used to distinguish isobaric cross-linked peptides with thiazine and thiosuccinimide linkers. Peptides that are cross-linked with thiazine linkers are very stable in MALDI and are thus detected only as (M+H)+ ions. On the other hand, cross-linked peptides with thiosuccinimide as the linker are clearly more susceptible to degradation by MALDI, and the thioether bond of thiosuccinimide is partially cleaved. As a result, in addition to the intact cross-linked peptides, the free thiol-containing peptides and peptides attached to the original N -propionyl succinimide, which constitute the conjugate, are clearly detected in the MALDI-MS spectra. This behavior was also observed for cross-linked peptides with hydrolyzed thiosuccinimide linkers.
MALDI-MS/MS analysis can more reliably distinguish isobaric cross-linked peptides with thiazine and thiosuccinimide as linkers. The former gives fragment ions derived from cleavage at the new pseudo-peptide bond produced by trans-cyclization in the thiosuccinimide linker14,15; the latter gives two types of the fragment ions due to 1,4-elimination that occurs in the gas phase (Figure 4). MALDI-MS or MALDI-MS/MS can be used as a simple technique to identify isobaric cross-linked peptides that are prepared via maleimide chemistry.