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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Beta-Aspartic Acid Impairs the Ability to Bind Metal Ions byImmunosuppresory Fragment of Ubiquitin and Other Peptidesas Studied by ESI-MS/MSMarek Cebrat, Marlena Zajączkowska, Piotr Stefanowicz,and Zbigniew SzewczukFaculty of Chemistry, University of Wrocław, Wrocław, 50-383, PolandIntroductionRecently ubiquitin was suggested as a promising anti-inflammatory protein therapeutic [1].We found that a peptide fragment corresponding to the ubiquitin 50–59 sequence(LEDGRTLSDY, Ubq 50-59 ) possessed the immunosuppressive activity comparable withthat of ubiquitin. The peptide was much less toxic than cyclosporine, particularly at higherdoses [2]. The cyclic peptide designed to mimic the ubiquitin 50–59 loop stronglysuppressed the immune response, suggesting that ubiquitin and its LEDGRTLSDYfragment may interact with the same hypothetical receptors [3].It is known that the formation of complexes of proteins and peptides with metal ionsoften plays a significant role in their interaction with the respective receptors [4]. Ourearlier studies indicated that the mass spectrometry can provide reliable information on thestoichiometry and a possible binding mode of the peptide-metal complexes [5,6].Therefore, we applied high-resolution mass spectrometry to study the interaction ofLEDGRTLSDY fragment analogues with Cu 2+ and Zn 2+ ions. A series of the peptideanalogues, which included acetylated and amidated peptides and sequences with Aspresidue replaced by beta-aspartic acid ( Asp), was synthesized. A model tetrapeptideH- Asp-His-Gly-His-OH as well as analogues of natural, biologically active peptides(angiotensin, AT; fibrinopeptide A, FPA; thymopentin, TP-5; osteonectin, SPARC) withAsp to Asp substitution were also synthesized and tested:Ubiquitin50-59 fragment analogues:H-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-OH Ubq 50-59Ac-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-OH AcUbq 50-59H-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-NH 2 Ubq 50-59 -NH 2Ac-Leu-Glu-Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-NH 2 AcUbq 50-59 -NH 2H-Leu-Glu- Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-OH [ Asp 3 ]Ubq 50-59Ac-Leu-Glu- Asp-Gly-Arg-Thr-Leu-Ser-Asp-Tyr-OH [ Asp 3 ]AcUbq 50-59Other peptides:H- Asp-His-Gly-His-OH [ Asp 1 ]DHGHH- Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH [ Asp 1 ]ATH-Ala- Asp-Ser-Gly-OH[ Asp 2 ]FPAH-Arg-Lys- Asp-Val-Tyr-OH[ Asp 3 ]TP-5Ac-Thr-Leu-Glu-Gly-Thr-Lys-Lys-Gly-His-Lys-Leu-His-Leu- Asp-Tyr-NH 2[ Asp 14 ]AcSPARC-NH 2Results and DiscussionPeptides were synthesized by a manual solid phase peptide synthesis using standard Fmocprotocol. Peptide solutions were mixed with the metal ions solutions (1:1) and incubatedfor 24 hours at 5°C before MS experiments. Interaction of the peptides with metal ionswere studied on Bruker apex ultra 7T FT-ICR mass spectrometer equipped with an ESIsource using standard instrument settings. Positive ions were analyzed.The analysis of ESI-MS spectra of the studied peptides containing Asp residueclearly shows that they are poor ligands for Cu 2+ and even worse for Zn 2+ ions. In a case ofthe LEDGRTLSDY peptide (Ubq 50-59 fragment) and its analogues the substitution of Asp 3residue by Asp 3 as well as N-terminal acetylation drastically reduces the ability to form apeptide-metal complex. For example, peaks of the Ubq 50-59 fragment complexed with Cu 2+ion is very intense (peaks at m/z 615.22 and 1229.43) whereas they are almost completelyabsent for [ Asp 3 ]Ubq 50-59 (Figure 1). C-terminal amidation does not influence binding ofCu 2+ ions.The presence of Asp residue in position three of the peptide chain (Ubq 50-59 fragmentanalogues and [ Asp 3 ]TP-5 peptide) completely prevents the formation of the complexes596