Smart peptide libraries are accessible via enzymatic modifications

The minireview summarizes the recent preparation of thefollowing unusually modified combinatorial peptide collectionsuseful for diagnostics and screening in drug finding. Tissuetransglutaminase catalyzes cross couplings with transamidationbetween Gln and Lys peptide chains resulting in libraries withisopeptide bonds. The enzyme is involved in the triggering ofautoantigenic B- and T-cell epitopes of coeliac disease. Themicrobial enzyme EpiD involved in lantibiotic biosynthesiscatalyzes oxidative decarboxylation of C-terminal cysteineresidues in peptide libraries transforming peptidyl-cysteinesto peptide (2-mercaptovinyl)amides. Novel backbone modifiedpeptide libraries are prepared using oxazole and thiazolebuilding blocks carrying amino acid side chains. These aminoacids have been found in many biologically active naturalproducts from marine and microbial organisms such as microcinB17. Dityrosine and isodityrosine linked peptide dimerlibraries are accessible by oxidative phenol coupling usinghorseradish peroxidase. Such structural elements are found forexample in the polycyclic glycopeptide antibiotics of thevancomycin type. Microstructured layers of linear and cyclicpeptide libraries are generated on transducer surfaces forcellular assays, sensor developments and even chiralrecognition. Examples include a light-directed andmicrostructured electrochemical polymerization of phenollabelled peptides.     

9th Annual Symposium on Advances in Separation Science and Mass Spectrometry

Evaluation of: Di Girolamo F, Boschetti E, Chung MC, Guadagni F, Righetti PG. ‘Proteomineering’ or not? The debate on biomarker discovery in sera continues. J. Proteomics 74(5), 589–594 (2011).

The combinatorial peptide ligand library in association with mass spectrometry can greatly enhance the dynamic range of the analysis of low- and very low-abundance proteins constituting the vast majority of species in any sample. When compared with untreated samples, the increment in detection of low-abundance species appears to be at least fourfold. Recently, the combinatorial peptide ligand library has been challenged; however, it has been clearly demonstrated in the evaluated paper that the protocols for elution of the captured polypeptides make the difference. Therefore, the solid-phase ligand library made of hexapeptides remains a promising and unique tool for biomarker discovery.     

Mapping epitopes of neutralizing monoclonal antibodies using phage random peptide libraries

Identification of protective determinants from microbial proteins is a necessary step in the rational design of subunit vaccines. We have previously used a synthetic peptide scan (Pepscan) assay to map a panel of eight neutralizing monoclonal antibodies (mAb; designated as C1.1 to C1.8) to a common motif sequence from Chlamydia trachomatis. In the present study, five of the eight mAbs were used to screen phage random peptide libraries. mAbs C1.1 and C1.3 selected a motif sequence of G-L-X-N-D from a pIII-based phage random peptide library and a motif sequence of G-X-X-N-D from a pVIII-based random peptide library while mAbs C1.6 to C1.8 failed to select recognizable motifs from either of the phage libraries. However, C1.6 to C1.8 bound to the same motif sequence displayed on phage when the appropriate conformational constraints were imposed onto the motif sequence. Thus the specificity of the mAbs identified on Pepscan assays correlates with the mAbs’ dependence on local epitope constraints displayed on the phage surface. Received 12 August 1996/ Accepted in revised form 03 November 1996     

Efficient methodology for the cyclization of linear peptide libraries via intramolecular S-alkylation using Multipin solid phase peptide synthesis.

Methodology is described here for the efficient parallel synthesis and cyclization of linear peptide libraries using intramolecular S-alkylation chemistry in combination with Multipin solid phase peptide synthesis (Multipin SPPS). The effective use of this methodology was demonstrated with the synthesis of a 72-member combinatorial library of cyclic thioether peptide derivatives of the conserved four-residue structural motif DD/EXK found in the active sites of the five crystallographically defined orthodox type II restriction endonucleases, EcoRV, EcoRI, PvuII, BamHI and BglI.     

Identification of monoclonal antibody defined epitopes on human leukocyte antigens utilizing phage display peptide libraries

Summary Utilizing phage display peptide libraries, we have identified and mapped the antigenic determinants recognized by mouse monoclonal antibodies (mAb) on two sets of immunologically important molecules, HLA class I and class II antigens. Anti-HLA class I mAb TP25.99 recognizes a conformational and a linear determinant on distinct regions of the HLA class I α3 domain. Anti-HLA class I mAb HO-4 recognizes a conformational determinant on the α2 domain of HLA-A2 and A28 allospecificities. Anti-HLA-DR1,-DR4,-DR6,-DR8,-DR9 mAb SM/549 recognizes a conformational determinant on the β chain of HLA class II antigens. These results indicate the versatility of phage display peptide libraries to characterize antigenic determinants recognized by anti-HLA mAb.     

Accuracy of side-chain prediction upon near-native protein backbones generated by ab initio folding methods

The ab initio folding problem can be divided into two sequential tasks of approximately equal computational complexity: the generation of native-like backbone folds and the positioning of side chains upon these backbones. The prediction of side-chain conformation in this context is challenging, because at best only the near-native global fold of the protein is known. To test the effect of displacements in the protein backbones on side-chain prediction for folds generated ab initio, sets of near-native backbones (≤ 4 Å Cα RMS error) for four small proteins were generated by two methods. The steric environment surrounding each residue was probed by placing the side chains in the native conformation on each of these decoys, followed by torsion-space optimization to remove steric clashes on a rigid backbone. We observe that on average 40% of the χ1 angles were displaced by 40° or more, effectively setting the limits in accuracy for side-chain modeling under these conditions. Three different algorithms were subsequently used for prediction of side-chain conformation. The average prediction accuracy for the three methods was remarkably similar: 49% to 51% of the χ1 angles were predicted correctly overall (33% to 36% of the χ1+2 angles). Interestingly, when the inter-side-chain interactions were disregarded, the mean accuracy increased. A consensus approach is described, in which side-chain conformations are defined based on the most frequently predicted χ angles for a given method upon each set of near-native backbones. We find that consensus modeling, which de facto includes backbone flexibility, improves side-chain prediction: χ1 accuracy improved to 51–54% (36–42% of χ1+2). Implications of a consensus method for ab initio protein structure prediction are discussed. Proteins 33:204–217, 1998. © 1998 Wiley-Liss, Inc.     

In situ genotyping of a pooled strain library after characterizing complex phenotypes

In this work, we present a proof‐of‐principle experiment that extends advanced live cell microscopy to the scale of pool‐generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single‐molecule fluorescence time‐lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.     

New targeted approaches for the quantification of data‐independent acquisition mass spectrometry

The use of data‐independent acquisition (DIA) approaches for the reproducible and precise quantification of complex protein samples has increased in the last years. The protein information arising from DIA analysis is stored in digital protein maps (DIA maps) that can be interrogated in a targeted way by using ad hoc or publically available peptide spectral libraries generated on the same sample species as for the generation of the DIA maps. The restricted availability of certain difficult‐to‐obtain human tissues (i.e., brain) together with the caveats of using spectral libraries generated under variable experimental conditions limits the potential of DIA. Therefore, DIA workflows would benefit from high‐quality and extended spectral libraries that could be generated without the need of using valuable samples for library production. We describe here two new targeted approaches, using either classical data‐dependent acquisition repositories (not specifically built for DIA) or ad hoc mouse spectral libraries, which enable the profiling of human brain DIA data set. The comparison of our results to both the most extended publically available human spectral library and to a state‐of‐the‐art untargeted method supports the use of these new strategies to improve future DIA profiling efforts.     

Large scale of identification of differentially expressed genes in the regenerating rat liver after SISPH

After partial hepatectomy (PH), the remnant paren-chyma can completely recover lost liver mass and function in about one week[1,2]. Although adult hepa-tocytes are normally quiescent, they are readily primed to pass from G0 to G1 phase within 2―6 h after PH. The first peak of DNA synthesis appears 24 h after PH, while cell division peaks at 36 h. The liver cells then enter a second cell cycle, and redifferentiation and reconstruction of structure and function[3―6] take place. A great nu…     

Design of a bivalent peptide with two independent elements of secondary structure able to fold autonomously.

This article describes a strategy to develop, starting from a de novo design, bivalent peptides containing two different (alpha-helix and beta-hairpin) and independent secondary-structure elements. The design was based on the use of conformationally restricted peptide libraries. Structural characterization by NMR revealed that the peptides were stable and did not show any long-range NOE interactions between the N-terminal beta-hairpin and the C-terminal alpha-helix. These results suggest that the two elements of secondary structure are stable and well folded.