HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides.

Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

Isotope or mass encoding of combinatorial libraries.

BACKGROUND: Combinatorial chemistry using solid-phase synthesis is a rapidly developing technology that can result in a significant reduction in the time required to find and optimize lead compounds. The application of this approach to traditional medicinal chemistry has led to the construction of libraries of small organic molecules on resin beads. A major difficulty in developing large combinatorial libraries is the lack of a facile encoding and decoding methodology to identify active compounds. RESULTS: Several encoding schemes are described which use the ability of mass spectrometry to ascertain isotopic distributions. Molecular tags are attached to resin beads in parallel or on the linker used for chemical library synthesis. The tags are encoded via a controlled ratio of a number of stable isotopes on the tagging molecules, and range from a single to a complex isotopic distribution. CONCLUSIONS: A novel coding scheme is described that is useful for the generation of large encoded combinatorial libraries. The code can be cleaved after assay and analyzed by mass spectrometry in an automated fashion. An important element of the combinatorial discovery process is the ability to extract the structure-activity relationship (SAR) information made available by library screening. The speed and sensitivity of the mass-encoding scheme has the potential to determine the full SAR for a given library.

The loss of female sex pheromone after mating in the corn earworm moth Helicoverpa zea: identification of a male pheromonostatic peptide.

Female moths often become depleted of sex pheromone after mating as the various components of virgin behavior are switched off. In examining a potential male contribution to these events in the corn earworm moth Helicoverpa zea, we have characterized a basic polypeptide from the tissues producing (accessory glands) and storing (duplex) the seminal fluids. The peptide evokes the depletion of sex pheromone when injected into virgin females. This pheromonostatic peptide (PSP) is 57 amino acids long and contains a single disulfide bridge. It is blocked at the N terminus with pyroglutamate and at the C terminus by amidation. As little as 23 ng of peptide evokes the near-complete depletion of pheromone in decapitated (neck-ligated) females that had been injected with pheromone biosynthesis-activating neuropeptide. Activity is approximately 15-fold less in intact virgins, showing that the head limits the expression of activity in these injected females. Females mated to surgically impaired males, capable of producing a spermatophore but not transferring spermatozoa or seminal fluids, are depleted of pheromone by injected peptide. Females whose abdominal nerve cords have been severed are not depleted of pheromone after mating. Thus, neural signals either descending or ascending via the nerve cord are required for the depletion of pheromone after mating. PSP, from the seminal fluids, may participate in this process by direct or indirect action on the glandular tissue; if so, it represents an unusual mechanism in insects for the regulation by seminal fluids of postmating reproductive behavior.

Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling.

Microcapillary HPLC electrospray ionization tandem mass spectrometry was used to sequence 15 peptides eluted from HLA-B7. Sequence alignment implicated four peptide positions in specific interactions with the class I molecule, and their importance was confirmed using synthetic peptides. Because no crystal structure for HLA-B7 was available, computer-assisted modeling was used to understand novel aspects of peptide binding specificity and to accurately predict the effect of defined changes in peptide structure. The results demonstrate that mass-spectrometric sequencing coupled with computer-assisted modeling can be used in the absence of a crystal structure to make accurate predictions concerning requirements for peptide binding to class I molecules. These techniques may be valuable to predict or engineer T cell epitopes.