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.