Cysteine-reactive covalent capture tags for enrichment of cysteine-containing peptides.

Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so-called cysteine-reactive covalent capture tags (C3T), for the isolation of Cys-containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine-containing peptides. The combination of C3T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins.

Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate.

The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and beta-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1' and P2'. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1' > P2 > P2'. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4'(dimethylamino)phenylazo]benzoyl-EVYAVES-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The approximately 4 x 10(6) M(-1) x s(-1) specificity constant of PrtA (at K(m) approximately 5 x 10(-5) M and k(cat) approximately 2 x 10(2) s(-1)) on this substrate was the highest activity for a serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.

The synthesis of alternative diketopiperazines as potential RGD mimetics.

Alternative RGD mimetics-with the exception of glycine-c(Arg-Asp) 1, c(Arg-Glu) 2 and c[Arg-Asp(Phe-OH)] 3 were synthesized. The DKPs were prepared on solid phase with orthogonal protection allowing further derivatization in solution. During solution phase cyclization in NH(3)/methanol, the side chain benzyl ester group of H-Arg(Tos)-Asp(OBzl)-OMe and H-Arg(Tos)-Glu(OBzl)-OMe suffer transesterification, while beta-t-butyl or beta-cyclohexyl esters are stable under the same conditions. In spite of the simple structure, all compounds bind selectively to the alpha(v)beta(3) integrin receptor, 3 showing the highest affinity with an IC(50) value of 0.74 microM value. On the other hand only 3 binds with measurable activity to the alpha(IIb)beta(3) receptor (IC(50) 159 microM). The binding affinities seem to be in accordance with the distances between the arginine guanidino and the aspartic acid carboxyl group in extended conformation determined by semiempirical geometry optimization.

Copper(II) interaction with unstructured prion domain outside the octarepeat region: speciation, stability, and binding details of copper(II) complexes with PrP106-126 peptides.

Copper(II) complexes of the neurotoxic peptide fragments of human and chicken prion proteins were studied by potentiometric, UV-vis, CD, and EPR spectroscopic and ESI-MS methods. The peptides included the terminally blocked native and scrambled sequences of HuPrP106-126 (HuPrPAc106-126NH2 and ScrHuPrPAc106-126NH2) and also the nona- and tetrapeptide fragments of both the human and chicken prion proteins (HuPrPAc106-114NH2, ChPrPAc119-127NH2, HuPrPAc109-112NH2, and ChPrPAc122-125NH2). The histidyl imidazole-N donor atoms were found to be the major copper(II) binding sites of all peptides; 3N and 4N complexes containing additional 2 and 3 deprotonated amide-N donors, respectively, are the major species in the physiological pH range. The complex formation processes for nona- and tetrapeptides are very similar, supporting the fact that successive deprotonation and metal ion coordination of amide functions go toward the N-termini in the form of joined six- and five-membered chelates. As a consequence, the peptide sequences investigated here, related to the neurotoxic region of the human PrP106-126 sequence, show a higher metal-binding affinity than the octarepeat fragments. In the case of the HuPrP peptide sequences, a weak pH-dependent binding of the Met109 residue was also detected in the 3N-coordinated complexes.

Solution state conformation and degradation of cyclopeptides containing an NGR motif.

In contrast to the RGD-peptides, head to tail cyclization of LNGRV and LNGRv caused only a marginal change in their integrin receptor affinity as shown by the limited effect and selectivity on the adhesion of endothelial cells to ECM components. Structure determination of the two cyclopeptides by NMR and MD, semiempirical and ab initio methods revealed that both are very flexible and take on multiple stable conformers in solution. This structural diversity, along with the presence of the Asn-Gly peptide bond, enhances succinimide ring formation leading to the hydrolysis of Asn. It has been demonstrated that c(LNGRV) suffers deamidation with time both in solution and during storage. As the isoaspartyl-peptide may co-elute with the asparginyl-peptide in the course of HPLC analysis, MS measurement is necessary to check the purity of peptides containing the NGR sequence. Our stability investigations raise the question whether the NGR motif or its hydrolysis product is effective in in vivo experiments.

Cell-physiological effects of elastin derived (VGVAPG)n oligomers in a unicellular model system.

Elastin is one of the most significant components of the extracellular matrix, which supports the stretchiness of the blood vessels via its helical structure and cross-links. Enzymatic decomposition of this protein could induce chemotactic responses of cell populations in the surrounding tissues by several peptide sequences, e.g. XGXXPG. In our present work the VGVAPG variant and its oligomers were studied. The objective of the experiments was to learn (i) whether the chemotactic effect of these peptides is general in different levels of phylogeny: (ii) whether increasing the number of monomer units influences the chemotactic behaviour of the cell? The trimer had the strongest chemoattractant effect in a wide concentration range (10(-12) - 10(-7) M), while the monomer and the pentamer were chemorepellent. All tri-, tetra-, penta- and hexamers could chemotactically select subpopulations with a high chemotactic responsiveness to the identical peptide, in the long term. With regard to its repellent effect, the pentamer had a negative effect on phagocytosis. All six oligomers had a growth-promoter effect in Tetrahymena. The characteristic cell-physiological effects of VGVAPG oligomers signal that molecules of the extracellular matrix can induce identical responses even in lower levels of phylogeny, e.g. in the Ciliates.

Synthesis of the C-terminal domain of the tissue inhibitor of metalloproteinases-1 (TIMP-1).

According to recent investigations, the C-terminal domain of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) is responsible for some biological effects that are independent of the enzyme-inhibiting effect of the N-terminal domain of the molecule. The C-terminal domain has been prepared for structure-biological activity investigations. After the chemical synthesis and the folding of the linear peptide. LC-MS and MALDI-MS analysis revealed that two isomers with different disulphide bond arrangements were formed. Since more than 30 folding experiments resulted in products with a very similar HPLC-profile, it was concluded that in the absence of the TIMP-1 N-terminal domain no entirely correct folding of the C-terminal domain occurred. Furthermore, it was observed that, in spite of several purification steps, mercury(II) ions were bound to the 6SH-linear peptide; it was demonstrated--using disulphide bonded TIMP-1(Cys145-Cys166) as a model--that mercury(II) ions can cause peptide degradation at pH 7.8 as well as in 0.1% trifluoroacetic acid.