Figures Legends:
Figure 1 . Possible cross-linking structures between Cys and Lys prepared by thiol-maleimide chemistry. Structure (I) : thiosuccinimide form; (II), (III) : Hydrolyzed forms of(I) , in which the five-membered thiosuccinimide ring was cleaved at “a” or “b”, respectively; (IV) : thiazine form, which is formed by the attack of the α-amino group of Cys on the carbonyl of the succinimide moiety and has the same molecular mass as(I) . The dashed square in Structure IV shows the new pseudo-peptide bond present in the thiazine ring.
Figure 2. Reverse-phase liquid chromatograms of the P1-P2 cross-linked peptide obtained after the synthesis (b) and incubation in 100 mM Tris/HCl buffer, pH=8.0 for 6 hours at 37oC (a) . The amino acid sequences of P1 and P2 are (A AAGGGAAAAK) and (C AAGGGAAAAK), respectively. The peaks marked with asterisks in both chromatograms correspond to the disulfide-linked P2-S-S-P2 homodimer formed as a by-product. The left and right Y-axes represent the absorbance (mV) at 210 nm of the upper and lower chromatograms, respectively. Numbers in red above each fraction indicate retention times.
Figure 3. MALDI-MS spectra of the cross-linked peptide, P1 (A AAGGGAAAAK)-P2 (C AAGGGAAAAK)(a) , its hydrolyzed (P1-P2)H (b) and thiazine (P1-P2)T (d) forms. The insets in (a) and(b) are the expanded views of the red dashed squares. The signals marked with I, II, III, and IV in the insets are assigned to the structures in (d) . The structures of P1-P2, (P1-P2)H and (P1-P2)T are depicted at the right side in (a) , (b) and (c) , respectively. P2-SH in the spectra correspond to the P2 peptide with a free Cys residue.
Figure 4. The 1,4-elimination observed for the cross-linked peptide containing thiosuccinimide. The two possible pathways are depicted in (a) and (b) . Pn denotes the peptides used in this study.
Figure 5 . MALDI-MS spectra of the cross-linked peptides, P2 (C AAGGGAAAK)-P4 (Ac-GANAPK EPQR) (a) and the thiazine form (P2-P4) T (b). The inset of (a) is the expanded view of the red dashed square. P2-SH corresponds to the P2 peptide with a free Cys residue. The signals marked with P4* in (a) correspond to the structures depicted in (c).
Figure 6 . MALDI-MS/MS spectra of the cross-linked peptides, P1-P2 (a) and (P1-P2)T (d). The areas enclosed by the dashed squares in (a) and (d) are enlarged in (b) and (c), respectively. The m/zvalues in green and red indicate fragment ions that are specifically observed for the thiosuccinimide and thiazine forms, respectively. “L” indicates the N -propionyl maleimide linker. The assignments of backbone-derived fragment ions are summarized in Figures S10-S11.