Tilmann Walk

Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins

The human plasma protein patterns obtained by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a good model system for post‐translational modifications because of the existence of several „ladders”︁ of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883—906], so‐called „trains”︁ of spots. Our investigation of several proteins, amongst others beta2‐microglobulin and the haptoglobin chains, found the differences in isoelectric points (pI) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2‐D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed‐phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine‐containing and deamidated asparagine‐containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2‐D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the „ladder”︁ is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.

NEW ADVANCES IN THE BIOSYNTHESIS OF GLYCOPEPTIDE ANTIBIOTICS OF THE VANCOMYCIN TYPE FROM AMYCOLATOPSIS MEDITERRANEI

Linear biosynthesis products from balhimycin, a tricyclic glycopeptide antibiotic of the vancomycin type, were isolated from mutants of Amycolatopsismediterranei. The structures of these intermediates, determined by NMR spectroscopy, give new impulses for the understanding of the vancomycin biosynthesis.

Solid phase synthesis of diverse isoxazolidines via 1,3-dipolar cycloaddition

Diverse substituted isoxazolidines 2–4 are assembled on solid phase by condensing hydroxylamines with aldehydes and trapping the resulting nitrones with various dipolarophiles. The one-pot three-component cycloaddition reaction yields the isoxazolidines in good to excellent yield. Three different reaction pathways - each dealing with one of the three components attached to the solid support - are evaluated with respect to versatility and yield of the synthesized isoxazolidines. Structures and relative configurations of representative isoxazolidines are elucidated with 1H-NMR, TOCSY, HSQC and NOESY. The reaction of polymer-bound hydroxylamines with aldehydes and alkenes is found to be the most successful. The kinetics of the solid phase reaction are determined by FT-IR-ATR spectroscopy. Polymer-bound nitrones are stable for the split/combine synthesis. A library of isoxazolidines is synthesized by the split/combine method and analyzed by means of HPLC-MS.

FT‐IR Mapping—A New Tool for Spatially Resolved Characterization of Polymer‐Bound Combinatorial Compound Libraries with IR Microscopy

In the search for selective receptors, a promising approach is provided by immobilization of cyclopeptides on sensor surfaces (see picture) and subsequent detection of low molecular weight analytes with reflectometric interference spectroscopy (RIfS). Together with combinatorial chemistry, this innovative method offers a great potential for applications as sensors.

Neues zur Biosynthese von Glycopeptid-Antibiotika des Vancomycin-Typs aus Amycolatopsis mediterranei

Lineare Biosyntheseprodukte von Balhimycin, einem tricyclischen Glycopeptid-Antibiotikum vom Vancomycin-Typ, konnten aus Mutanten von Amycolatopsismediterranei isoliert werden. Die NMR-spektroskopisch aufgeklarten Strukturen dieser Intermediate geben neue Impulse fur das Verstandnis der Vancomycin-Biosynthese.

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition.

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.

Increased HLA-DQ2-affinity of a synthetic gliadin peptide by acid-induced deamidation of glutamine residues

Presentation of antigenic gliadin peptides by the HLA-DQ2 molecule is considered as a key event in celiac disease pathogenesis. Chemical deamidation of the side chains of glutamine residues might have a strong influence on gliadin peptide binding to the DQ2 molecule. Glutamine deamidation of A-gliadin peptide (45-56) under acidic conditions corresponding to the gastric environment was studied using RP-HPLC, Edman degradation, capillary electrophoresis and electrospray mass spectrometry. Deamidation resulted in peptides with increased DQ2-affinities as assessed in a cell-free binding assay.

Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry.

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.

ESI-Fourier-Transform-Ionenzyklotronresonanz-Massenspektrometrie (ESI-FT-ICR-MS) – schnelle höchstauflösende Analytik für kombinatorische Verbindungsbibliotheken

Die direkte Bestimmung der Elementarzusammensetzungen der Komponenten von Verbindungskollektionen aus der kombinatorischen Chemie wird durch die ESI-FT-ICR-Massenspektrometrie ermoglicht. Die Kopplung mit HPLC erschliest eine weitere Dimension hochstauflosender aussagekraftiger Analytik. Dargestellt ist die im Vergleich zur Quadrupol-MS erheblich verbesserte Auflosung der ESI-FT-ICR-MS bei Messungen einer durch Festphasensynthese erhaltenen Verbindung.