Hedvig Medzihradszky-Schweiger

A simple method for monitoring the cysteine content in synthetic peptides.

A new method for the determination of the cysteine content of synthetic peptides is described. For this purpose the classical amino acid analysis is not suitable, as cysteine may undergo oxidative dimerisation to cystine during the hydrolysis or evaporation. The intact peptide was reacted with N‐ethylmaleimide (NEM) yielding a stable S‐(N‐ethylsuccinimido)‐cysteine derivative, which could be separated by RP‐HPLC and characterised by mass spectrometry. For the quantitative determination less than 0.1 µM peptide was sufficient. Copyright

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of alpha-melanotropin, [Glp5]alpha-MSH(5-10), [Glp5,D-Phe7]alpha-MSH(5-10), [Sar5,D-Phe7]alpha-MSH(5-10), [Nle4,D-Phe7]alpha-MSH(4-10), [N-carbamoyl]alpha-MSH(5-10), and formyl and acetyl derivatives of alpha-MSH(5-10), [Gly5]alpha-MSH(5-10) and [Gly5,D-Phe7]alpha-MSH(5-10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.

Analysis and Characterization of Combinatorial Mixtures of Mucin-2 Antigen Peptides

An epitope motif, TX1TX2T, of mucin-2 glycoprotein was identified by means of a monoclonal antibody, Mab 994 raised against a synthetic mucin-derived 13-mer pep-tide-conjugate [1,2]. For determination of the epitope sequence recognised with highest affinity by the antibody, a combinatorial approach was applied. We have prepared 19 sub-libraries (TATX2T, TDTX2T, TYTX2T) with portioning-mixing technique [3] using Boc/Bzl chemistry. Each library contained one pre-selected amino acid in position X1 and all proteinogenic amino acids, except Cys, in position X2 [1]. Antibody binding of sub-libraries was most profound when Gin was at the X1 position. 19 individual peptides corresponding to the TQTX2T sub-library were synthesised and tested. Binding data showed that apart from the native TQTPT peptide other compounds (e.g., TQTAT) were also recognised [4]. For the analytical characterization of the TQTX2T sub-library MALDI-TOF-MS and ultrahigh-resolution ESI-FTICR-MS as well as amino acid analysis were applied (cysteine was omitted for practical reasons). Shorter libraries corresponding to X2T and TX2T were also produced for amino acid analysis. To evaluate the resolution power of these MS methods we have prepared and tested “artificial” peptide mixtures of different complexity by mixing 6, 10, or 19 individual peptides corresponding to the TQTX2T. Thus equimolar mixtures of penta-peptides were analysed and compared with the combinatorial mixture.