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Guanylin 99-115 is a small human peptide hormone with two disulfide bonds. The four cysteinyl residues in this peptide allow the formation of two disulfide bridges in three different ways but only the 1-3/2-4 combination is able to bind to the receptor and cause an increase of intracellular cGMP, while the other isomers are biologically inactive. Using guanylin 99-115 as a model peptide, the aim of this study was to investigate whether it is possible to distinguish the differently bridged isomers directly by tandem mass spectrometry. Guanylin isomers were generated by performing an air oxidation of fully reduced guanylin 1-3/2-4 obtained by chemical synthesis. The reaction product is a mixture of guanylin 1-4/2-3 and guanylin 1-2/3-4 in a ratio of 3:1, but there is virtually no guanylin 1-3/2-4. The two biologically inactive peptides were separated by reversed-phase high pressure liquid chromatography (HPLC). Using low-energy collision-induced dissociation tandem mass spectrometry, it was possible to distinguish unambiguously between the three guanylin isomers. This was possible due to the identification of a large number of fragments with intact disulfide bonds. Accordingly, this strategy of a direct and sensitive analysis should work as well for other peptides, with the potential to determine an undefined disulfide bond pattern.

A method for the mapping of peptide mixtures, heterogeneous with respect to the concentration and the size of individual peptides, was established with the aim of obtaining a comprehensive analysis of human urine peptides. Peptide extraction and fractionation were optimized to achieve a two-step analysis, using reversed-phase and ion-exchange chromatography. Highly sensitive detection of peptides was performed by coupling microbore HPLC with electrospray mass spectrometry (ESI-MS). Peptides such as urodilatin, angiotensin and fragments of psoriasin, granulin and uromodulin were isolated and sequenced. The procedure presented here is a tool for the analysis of complex peptide mixtures from human urine.

The contractile action of bradykinin on isolated smooth muscles is known to be potentiated by special peptides isolated from snake venoms and other animal sources. A fraction F (AppF) has been derived from Agkistrodon piscivorus piscivorus venom. This fraction does not increase the depressor effect of bradykinin n blood pressure, potentiates the action of bradykinin on isolated guinea-pig ileum and prolongs the duration of relaxation in isolated rat duodenum. This fraction was able to inhibit the conversion of angiotensin I to angiotensin II in vitro. The primary structure of this decapeptide is given.

Here we report the isolation and characterization of an antibacterial peptide from bovine milk inhibiting the growth of Escherichia coli, and Staphylococcus carnosus. The primary structure of the peptide was revealed as a 39-amino-acid-containing fragment of bovine alpha s2-casein (position 165-203) by means of Edman amino acid sequencing and mass spectrometry. Since human milk does not contain any casein-alpha s2, these findings could explain the different influence of human and bovine milk on the gastrointestinal flora of the suckling.

Based on the global phosphorylation approach, a selective synthesis of (Ser10-phosphatidyl)-urodilatin (phosphourodilatin), which contains 32 amino acid residues and a disulfide loop is described. The peptide was assembled stepwise on a polyethyleneglycol-polystyrene support using Fmoc-chemistry. The phosphorylation was performed on-resin by phosphitylation with a large excess of di-tert-butyl-N,N-diethylphosphoramidite within 1 hour, followed by oxidation with tert-butylhydroperoxide to the protected phosphopeptide. After cleavage and deprotection the disulfide bridge was introduced without side reactions by iodine titration of the mono-acetamidomethyl protected crude peptide. During the synthetic pathway, the acylation with side chain-unprotected Fmoc-serine and the phosphitylation satisfactorily yielded the expected intermediates. In some phosphorylation experiments a by-product having a reduced mass corresponding to the H-phosphonate was observed. Illustrated with the synthesis of phosphourodilatin, this type of by-product, which could not be separated by HPLC, and the difficult amino acid sequence make the synthesis of a large phosphopeptide a more delicate task than the synthesis of short phosphopeptides, which do not contain oxidation-sensitive amino acids, difficult sequences or additional structural elements such as disulfide loops. The biological activity of phosphourodilatin was compared with non-phosphorylated urodilatin in two assay systems. Both peptides revealed a vasorelaxant effect on aortic smooth muscle strips and induced a cGMP-generation in RFL-6 cells with increasing dose dependency.

Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites. Exposure of the intact murine red cell to chymotrypsin leads to the formation of two fragments of 67 kDa and 41 kDa, which are equivalent to the 60 kDa and the 35 kDa fragments of the human band 3. Internal trypsin cleaves the chymotryptic 67 kDa fragment while the 41 kDa fragment appears essentially unaffected. The 67 kDa fragment is first degraded to 64 kDa, then further to 22 kDa and finally to 19 kDa. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) combines with murine band 3 protein as it does with human band 3. Anion transport is maximally inhibited when 5.10(5) H2DIDS molecules per cell are bound to band 3. As in the human red cell, after exposure to high pH (9.0-9.5) of the H2DIDS-labeled, chymotryptically cleaved band 3 intramolecular cross-linking takes place. This joins the 67 and 41 kDa chymotryptic pieces together to form a peptide of the original molecular mass of band 3 of 108 kDa. If cross-linking is performed after additional tryptic cleavage, the 19 and 22 kDa pieces join together with 41 kDa pieces to form overlapping bands that cover the molecular weight range from 60 to 63 kDa.