Differentiation by MALDI-MS and -MS/MS analysis of cross-linked peptides with thiosuccinimide and thiazine linkers.
The MALDI-MS spectra of cross-linked peptides (P1-P2) and (P2-P4) with a thiosuccinimide linker were compared with their corresponding isomers with a thiazine linker. Both of the cross-linked peptides with the thiosuccinimide linker gave a signal at m/z 847 which corresponds to the thiol-containing peptide (P2-SH). Their counterparts, linked to the N -propionyl maleimide or N -propionyl succinimide, were observed at m/z 966.49 and 968.50 (inset of Figure 3a), and atm/z 1260.32 and 1262.33 (inset of Figure 5a), which were assigned to P1-(I) and P1-(II) in Figure 3d, and P4-(I) and P4-(II) in Figure 5c, respectively. As in the case of P1* in the (P1-P2) cross-linked peptide (see Figure 3a), the P4 peptide was observed as N -propionyl maleimide or N -propionyl succinimide forms (Figure 5c), which arose from 1,4-elimination or reductive cleavage, respectively, at the C-S linkage between the Cys residue and the succinimide during the MALDI process.
The cross-linked peptides, P1-P2 and P2-P4, gave significant amounts of the isobaric thiazine forms under coupling conditions (Figure 2 and Figure S4) and are denoted as (P1-P2)T and (P2-P4)T. As described above, this transformation from the thiosuccinimide linker predominantly occurs in a spontaneous manner for the case where a conjugated peptide contains an N -terminal Cys. The molecular mass of the thiazine form was the same as that of the original thiosuccinimide form, but none of the fragment ions characteristic of the peptide with a thiosuccinimide linker were observed (Figures 3c and 5b). This result suggests that the thiazine linker is stable and resistant to cleavage during MALDI while the thiosuccinimide linker or its hydrolysis form is amenable to cleavage at the C-S linkage of the linker (Figures 3a and 5a).
These results suggest that the thiosuccinimide forms, including those that had been hydrolyzed, can be unambiguously identified by observing the peptides constituting the conjugate simultaneously in MALDI-MS (see Figures 3a, 3b, 5a, and S7-S9). In contrast, the thiazine form does not yield these peptide ions (Figures 3c and 5b). This suggests that the thioether bond, embedded in the six-membered ring of thiazine, is not susceptible to such cleavage of the conjugate that yields the two constituent peptides.
MALDI-MS/MS was then examined in an attempt to distinguish these isobaric peptides. The MALDI-MS/MS spectra of (P1-P2) with thiosuccinimide and (P1-P2)T with the thiazine linker were nearly identical, with respect to the m/z values of the backbone fragment ions for the P1 and P2 peptides (Figures 6a and 6d, Figures S10 and S11). However, there was a marked difference in the expanded region (m/z 792-1013, Figures 6b and 6c). The thiosuccinimide form (P1-P2) yielded two pairs of complementary fragment ions that were produced via a 1,4-elimination: Cys-SH and maleimide (Figure 4a), and dehydro-alanine and thiosuccinimide (Figure 4b), which was distinctly different from those produced via reductive cleavage in MALDI-MS (see Figure 3a). The other cross-linked peptides, (P1-P3) and (P3-P4), with the thiosuccinimide linker produced fragment ions that were similar to those observed for (P1-P2) (see Figures S12 and S13).
In the case of (P1-P2)T, the thiazine form, which is formed primarily when a cysteinyl peptide is cross-linked to another peptide containing Lys by BMPS (upper chromatogram in Figure 2, retention time 5.10 min), no ions were observed when the above C-S bond was cleaved. Instead, a pair of ions at m/z 886.36 (P1+71) and its counterpart of ion at m/z 927.30 (P2+L-71, where L denotes ‘linker’), highlighted in red, were newly observed (Figures 6c and 6d). These ions, specifically observed for (P1-P2)T, were those produced when cleaved at the new pseudo-peptide bond generated in the thiazine-containing linker (Figure 1, structure (IV)). Similar results were also obtained for (P2-P4)T (see Figures S14 and S15).
The above results demonstrate that MALDI-MS/MS allowed both isobaric cross-linked peptides with thiosuccinimide and thiazine linkers to be clearly differentiated, based on the fragment ions produced via a 1,4-elimination at the thiosuccinimide/Cys residue and the fragmentation of the newly formed pseudopeptide bond in a thiazine linker, respectively, the latter of which has also been observed by LC-MS/MS using HCD as a fragmentation method 15.