From spots to beads-PTM-peptide bead arrays for the characterization of anti-histone antibodies.

Antibodies that recognize PTMs of histones play a central role in epigenetic proteomic research. Modification-specific antibodies are employed in chromatin immunoprecipitation, for Western blotting and during the immunoprecipitation steps for MS-based global proteomic analyses. Knowledge about the antibodies' off-target binding is essential for the interpretation of experimental data. To address this challenge we developed a fast and cost efficient system for generating peptide bead arrays. We employed this method to establish a bead-based peptide array containing 384 peptides displaying phosphorylated, acetylated, methylated, and citrullinated N-terminal regions of histones H2A, H2B, H3 and H4 and controls. We profiled the binding of 40 PTM-specific antibodies important for epigenetic proteomic research.

Chemical labeling strategy with (R)- and (S)-trifluoromethylalanine for solid state 19F NMR analysis of peptaibols in membranes.

Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints theta by solid state (19)F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles tau and rho from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an alpha-helix, tilted away from the membrane normal by 8 degrees, and assembled as an oligomer. The new (19)F NMR label CF(3)-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.

Application of mixed peptide arrays to study combinatorial readout of chromatin modifications.

The N-terminal tails of histone proteins are massively decorated with post-translational modifications (PTMs), which play important roles in the regulation of gene expression. Several highly conserved chromatin interacting proteins can bind to histone modifications in a sequence and modification specific manner employing specific reading domains. These proteins often contain several reading domains, which can cooperate in the readout of different PTMs. To gain a better insight into the combinatorial readout of PTMs, we developed a method to study the binding of double reading domains to mixed peptide arrays containing two different peptides in each spot. For that, differently modified and unmodified peptides were prepared by SPOT synthesis and solubilized. Then, two peptides were mixed and spotted onto a glass slide creating peptide spots presenting two modifications on two different peptides. Different combinations of mixed spots containing modified and unmodified peptides were generated and incubated with recombinant double reading domains to study their synergistic binding. For validation of the method, we used the well-studied BPTF subunit of the NURF chromatin-remodeling complex. BPTF contains a plant homeodomain finger (PHD) and a Bromodomain recognizing H3K4me3 and H4K16ac, respectively. We first confirmed with peptide arrays and Fluorescence Anisotropy (FA) measurements that the BPTF PHD-Bromo (PB) domain interacts specifically with the expected modifications. Using our novel tool, we observed a strong and synergistic binding only to peptide spots containing both modifications, which was lost if one of the domains was inactivated by a mutation. These data indicate that BPTF-PB simultaneously interacts with both target modifications using its PHD and Bromodomain. In agreement with the synergistic peptide interaction on mixed peptide arrays, we also show that chromatin pulldown by BPTF-PB depends on the activity of both reading domains. We conclude that mixed peptide spot arrays are a powerful, cheap and novel method for screening the combinatorial interaction space of multidomain reading proteins. Using this approach hundreds of mixed peptide spots can be prepared and tested for binding in principle allowing for an unbiased medium throughput investigation.

Peptide arrays for development of PDGFRß Affine molecules.

PURPOSE: Peptide arrays represent an attractive method for identification of amino acid motifs that bind to target structures. Spotting derivatives of the linear peptide platelet-derived growth factor receptor (PDGFR)-P1, which has been identified to bind the extracellular domain of the platelet-derived growth factor receptor beta, allows the synchronous investigation of the target affinity of numerous ligands. PROCEDURES: A peptide array randomizing PDGFR-P1 was constructed by replacement of each amino acid by all 20 natural amino acids. Incubation of the array with PDGFRß and fibroblast growth factor receptor as negative control target was performed. Selected derivatives and fragments of PDGFR-P1 were chemically synthesized, radiolabeled, and evaluated in cell-based assays, using human pancreatic carcinoma BxPC3 and human breast cancer MCF7 cells. RESULTS: Binding capacity was increased for the derivate yG2 by exchange of 7S to 7R. Competition experiments demonstrated a binding decrease with increasing competitor concentration. Serum stability of yG2 was improved compared to the native ligand. CONCLUSION: Peptide arrays were successfully applied for the improvement of the PDGFRß binding peptide PDGFR-P1.