Fig. . Expanded regions (m/z 804-1034) in (a) and (b) correspond to the MALDI-MS/MS spectra of type 2 peptide (P1-P2) H with the hydrolyzed thiosuccinimide linker acquired by using and not using collision gas, respectively. The nomenclature and the proposed structures for the observed fragment derived from the linker are in agreement with previous manuscript . The fragment ion represented as P2= corresponds to P2 peptide with the N-terminal Cys residue transformed as a dehydroalanine residue. The fragment ion assigned as b 10β +H 2 O present in all MS/MS spectra corresponds to a C-terminal metastable fragmentation of the (M+H) + parent ions as described previously . The fragment ions y 3α and y 10α are originated by a favorable y n fragmentation at the N-terminal of the two proline residues present in the sequence of P4 peptide . The fragment ion indicated as P4 correspond to a fragmentation of the pseudopeptide bond between the lysine residue in P4 peptide and the linkers, either thiazine or hydrolyzed thiosuccinimide. The fragment ions labeled with an empty and filled circles correspond the P4+169 and P4+203, respectively . The fragment ions labeled with an empty and filled stars correspond the reduced P2 peptide (P2-SH) and also to the P2 peptide with the Cys residue transformed as a dehydroalanine, respectively . The nomenclature of the fragment ions is in agreement with the proposed by Schilling et. al. for the crosslinked peptides . The legend in (e) indicates the assignment as well as the m/z of the corresponding ions. O present in all MS/MS spectra corresponds to a C-terminal metastable fragmentation of the (M+H) + parent ions as described previously . The fragment ions labeled with an empty and filled circles correspond the P1+169 and P1+203, respectively . The fragment ions labeled with an empty and filled stars correspond the reduced P3 peptide (P3-SH) and also to the P3 peptide with the Cys residue transformed as a dehydroalanine, respectively . The structures of these fragment ions are the same shown in ) correspond to a Cterminal metastable fragmentation of the (M+H) + as described previously . The linker fragment ions labeled with an empty and filled circles in both MS/MS spectra correspond the P4+169 and P4+203 ions, respectively as indicated in (c). The fragment ions labeled with an empty and filled stars correspond the reduced P3 peptide (P3-SH) and also to the P3 peptide with the Cys residue transformed as a dehydroalanine, respectively . The structures of these fragment ions are the same shown in 18 Olabeled (P3-P4) H at the new carboxyl group generated upon hydrolysis of five-membered ring in a buffer prepared with 30 % (v/v) of H 2 18 O. Backbone fragment ions, either the b n and y n , and linker fragment ions highlighted in bold characters showed in the MALDI-MS/MS an isotopic ion distribution that evidenced the incorporation of 18 O in their structures. A red asterisk indicates in the structure of (P3-P4) H the position where the 18 O was incorporated. In (P3-P4) H the alpha and beta peptides are P4 and P3, respectively. To simplify the figure the structure of only one of the two positional isomers of the hydrolyzed thiosuccinimide linker was represented. 18 Olabeled (P2-P4) H at the new carboxyl group generated upon hydrolysis of five-membered ring in a buffer prepared with 30 % (v/v) of H 2 18 O. Backbone fragment ions, either the b n and y n , and linker fragment ions highlighted in bold characters showed in the MALDI-MS/MS an isotopic ion distribution that evidenced the incorporation of 18 O in their structures. A red asterisk indicates in the structure of (P2-P4) H the position where the 18 O was incorporated. The alpha and beta peptides are P4 and P2, respectively. To simplify the figure the structure of only one of the two positional isomers of the hydrolyzed thiosuccinimide linker was represented. 18 Olabeled (P1-P2) H at the new carboxyl group generated upon hydrolysis of five-membered ring in a buffer prepared with 30 % (v/v) of H 2 18 O. Backbone fragment ions, either the b n and y n , and linker fragment ions highlighted in bold characters showed in the MALDI-MS/MS an isotopic ion distribution that evidenced the incorporation of 18 O in their structures. A red asterisk indicates in the structure of (P1-P2) H the position where the 18 O was incorporated. The alpha and beta peptides are P1 and P2, respectively. To simplify the figure the structure of only one of the two positional isomers of the (P1-P2) H was represented. a) Fragment ions highlighted in blue were assigned manually by considering the cleavability of the linker fragmentation and their derived fragment ions. Z corresponds to a N-terminal acetylated cysteine residue. In the structure of the type 2 peptide, the α and β peptides are ( 1 Z-K 16 ) and ([ 192 T-K 200 ], respectively. The nomenclature of the backbone fragment ions is in agreement with the reported by Schilling et al . The linker fragment ions assigned as ( 1 Z-K 16 )+98) 1+ and ([ 192 T-K 200 ]+71) 1+ correspond to the α peptide and β peptides, containing the formerly crosslinked Lys and Cys modified by structures that increase their molecular mass by 98 Da and 71 Da, respectively. The linker fragment ion assigned as ( 1 Z-K 16 )+98) 1+ contain the Cys 1 S-alkylated by a succinic anhydride residue. The linker fragment ion assigned as ([ 192 a) Fragment ions highlighted in blue were assigned manually by considering the cleavability of the linker fragmentation and their derived fragment ions. Z corresponds to a N-terminal acetylated cysteine residue. In the structure of the type 2 peptide, the α and β peptides are ( 1 Z-K 16 ) and ([ 192 T-K 200 ], respectively. The nomenclature of the backbone fragment ions is in agreement with the reported by Schilling et al . The linker fragment ions assigned as ( 1 Z-K 16 )+98) 1+ and ([ 192 T-K 200 ]+71) 1+ correspond to the α peptide and β peptides, containing the formerly crosslinked Lys and Cys modified by structures that increase their molecular mass by 98 Da and 71 Da, respectively. The linker fragment ion assigned as ( 1 Z-K 16 )+98) 1+ contain the Cys a) Fragment ions highlighted in blue were assigned manually by considering the cleavability of the linker fragmentation and their derived fragment ions. Z corresponds to a N-terminal acetylated cysteine residue. In the structure of the type 2 peptide, the α and β peptides are ( 1 Z-K 16 ) and ([ 192 T-K 200 ], respectively. The nomenclature of the backbone fragment ions is in agreement with the reported by Schilling et al . The linker fragment ions assigned as ( a) Fragment ions highlighted in blue were assigned manually by considering the cleavability of the linker fragmentation and their derived fragment ions. Z corresponds to a N-terminal acetylated cysteine residue. In the structure of the type 2 peptide, the α and β peptides are ( 1 Z-K 16 ) and ([ 192 T-K 200 ], respectively. The nomenclature of the backbone fragment ions is in agreement with the reported by Schilling et al . The linker fragment ions assigned as ( 1 Z-K 16 )+98) 1+ and ([ 192 T-K 200 ]+71) 1+ correspond to the α peptide and β peptides, containing the formerly crosslinked Lys and Cys modified by structures that increase their molecular mass by 98 Da and 71 Da, respectively. The linker fragment ion assigned as ( 1 Z-K 16 )+98) 1+ contain the Cys 16 )+98) 1+ a) Fragment ions highlighted in blue were assigned manually by considering the cleavability of the linker fragmentation and their derived fragment ions. Z corresponds to a N-terminal acetylated cysteine residue. In the structure of the type 2 peptide, the α and β peptides are ( 1 Z-K 16 ) and a) Fragment ions highlighted in blue were assigned manually by considering the cleavability of the linker fragmentation and their derived fragment ions. Z corresponds to a N-terminal acetylated cysteine residue. In the structure of the type 2 peptide, the α and β peptides are ( 1 Z-K 16 ) and ([ 192 T-K 200 ], respectively. The nomenclature of the backbone fragment ions is in agreement with the reported by Schilling et al . The linker fragment ions assigned as ( 1 Z-K 16 )+98) 1+ and ([ 192 T-K 200 ]+71) 1+ correspond to the α peptide and β peptides, containing the formerly crosslinked Lys and Cys modified by structures that increase their molecular mass by 98 Da and 71 Da, respectively. The linker fragment ion assigned as ( 1 Z-K 16 )+98) 1+ contain the Cys