MALDI-MS analyses of cross-linked peptides with a thiosuccinimide linker and their hydrolyzed variants.
Considering the integrity of structural variants (hydrolyzed and thiazine forms17,18) of the linker moiety, which are relevant to the conjugation modes, four types of cross-linked peptides were prepared (Table 1). P3-P4 and P1-P3 gave only the hydrolyzed forms upon incubation under alkaline conditions (see Materials and Methods). P1-P2 and P2-P4 both gave the hydrolyzed and thiazine forms, the latter of which was the product of transcyclization between theN -terminal Cys of P2 and succinimide (see Figure 1).
The rp-HPLC profile of the P1-P2 cross-linked peptide showed a major fraction at 4.86 min (see the lower chromatogram in Figure 2). MALDI-MS analysis of this fraction (Figure 3a) confirmed the identity of this molecule. Intense signals at m/z 1812.88 and m/z 906.96 were assigned to (M+H)+ and (M+2H)2+ions of this cross-linked peptide with the thiosuccinimide linker, respectively (Table 1).
The signals at m/z 847.43 and m/z 968.50 had an appreciable intensity and were assigned to the P2 peptide with a free thiol and the P1 peptide linked to an N -propionyl succinimide, respectively (Figure 3a, structure (II) in 3d), which were confirmed by MS/MS analysis (Figures S5 and S6a) and were most likely produced via reductive cleavage during the MALDI procedure. In the MALDI-MS analysis of the crosslinked peptides prepared with other crosslinker reagents based on similar maleimide-thiol chemistry, signals resulting from cleavage of the thioether bond were also observed19,20.
Note that an expanded region of this MALDI-MS spectrum (see inset Figure 3a) also showed the presence of an ion at m/z 966.49, which corresponds to the P1 peptide, but with an N -propionyl maleimide group (structure (I) in Figure 3d). This fragment ion could be generated via a 1,4-elimination reaction at the thioether bond as depicted in Figure 4a, which was also observed by MS/MS analysis (Figure S6b).
The P1-P2 peptide but with a hydrolyzed linker, referred to as (P1-P2)H (rt. 4.70 min in the upper chromatogram, Figure 2), was prepared by incubating the original P1-P2 peptide (rt. 4.86 min in Figure 2, lower chromatogram) at pH=8.0 for 6 hours at 37oC. Its molecular mass was observed at m/z1830.64 (Figure 3b), the mass of which was larger by 18 Da than the original one (Figure 3a), suggesting that either of the two imide bonds in the thiosuccinimide had been hydrolyzed by the hydrolytic process (see structure (I) in Figure 1). It is noteworthy that the signals atm/z 847.32, 984.36 and 986.38 in Figure 3b could be assigned to the P2 peptide with a thiol and the P1 peptide with the counterpart but with the hydrolyzed linker (structure (III) and (IV) in Figure 3d), which were likely produced via 1,4-elimination or reductive cleavage, respectively, as observed for (P1-P2).
The above results clearly suggest that the linkage between the succinimide ring and Cys could be cleavable upon MALDI, the fact of which is also supported by the results obtained for the other cross-linked peptides, three of them with the thiosuccinimide linker (P1-P3), (P2-P4) and (P3-P4), and their corresponding analogs with a hydrolyzed thiosuccinimide linker, (P1-P3)H, (P2-P4)H and (P3-P4)H (see Figures S7-S9).
Considering the strong absorption of laser energy by hot matrices such as α-CHCA in MALDI and the subsequent transfer of energy to the analyte, it can be assumed that the excess energy stored in the analyte ions led to the spontaneous dissociation of the thioether bond, which is a relatively weak chemical bond. This result is consistent with the MALDI ionization method and suggests that thioether-based linkers are cleavable when the MALDI ionization method is used in such an analysis, and consequently provides useful information concerning the components of the cross-linked peptide.