ESI-MS studies on novel liquid homo-peptide libraries and their conjugate libraries directed by phosphorus oxychloride

Summary The reactions of natural non-polar amino acids with phosphorus oxychloride were investigated. Some non-polar amino acids, such as L-Phe, L-Leu and L-Val were treated with phosphorus oxychloride, then quenched with water or some bioactive compounds (L-menthol, cinchonidine and benzylamine), and yielded the corresponding peptide and peptide conjugate libraries. ESI-MS/MS was used to study the products of the reaction and confirm the structure of the library. This paper reports a simple method to synthesize the homo-oligo-peptide libraries and peptide conjugated libraries.     

Identification of HLA-Cw6.02 and HLA-Cw7.01 allele-specific binding motifs by screening synthetic peptide libraries

Unlike HLA-A and HLA-B, few peptide epitope motifs have been reported for HLA-C molecules. However, a number of cytotoxic T-lymphocyte epitopes derived from tumor antigens that bind to HLA-C molecules have been described. Here we report peptide-binding motifs for both HLA-Cw6.02 and HLA-Cw7.01 molecules. Recombinant human HLA molecules were generated and used to screen combinatorial 9mer peptide libraries. Complexes of HLA molecules properly folded and associated with ₂-microglobulin and peptides were identified using a conformation-specific HLA class I antibody conjugated to alkaline phosphatase. In the presence of substrate, peptide beads can be readily isolated and microsequenced to determine peptide identity. Of the peptides that bound to HLA-Cw6.02 and HLA-Cw7.01, 19 and 18 peptides, respectively, were sequenced, allowing motif identification for each C allele. This is the first report of an HLA-Cw7.01 peptide motif and extends the findings of Falk et al. [(1993) Proc Natl Acad Sci USA 90:12005] for an HLA-Cw6.02 motif. Anchoring amino acids for the HLA-Cw6.02 motif were phenylalanine or tyrosine in position (P)1, arginine in P2, and an aliphatic/aromatic residue at P9. Anchoring residues for HLA-Cw7.01 were positively charged amino acids in P1 and P2. Unlike most other HLA molecules, we were unable to assign P9 an anchoring residue, and we suspect that HLA-Cw7.01 binds peptides in an unconventional manner. Additionally, preferred amino acids were identified for both molecules. Identification of HLA-Cw6.02 and HLA-Cw7.01 peptide-binding motifs makes a significant contribution to the C allele peptide-binding motifs and will allow investigators to predict, design, and test HLA-Cw6.02 and HLA-Cw7.01 engineered peptides for immunotherapy.     

一种构建高质量随机肽库的有效方法

为构建完全随机化的基因工程肽库 ,克服现有简并方案中终止密码子和序列组成偏歧的不足 ,提出了一种新的简并DNA文库合成方式。通过这种分组式合成方式构建的肽库可以避免终止密码子的出现和氨基酸组成偏歧的发生 ,还可以控制随机化过程中不同氨基酸的参入比例。以一个 13肽库的合成过程为例对分组式合成法进行了实验 ,测序结果和对 19种氨基酸出现频率的统计表明没有终止密码子和半胱氨酸密码子出现 ,各氨基酸的出现频率接近均值 ,表明这种分组 混合 分组 ,辅以简并合成的方法是行之有效的 ,能满足各类高容量基因工程随机肽库要求。     

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO₂)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Straight-chain non-polar amino acids are good helix-formers in water

For comparison with earlier data on naturally occurring non-polar amino acids (Ala, Leu, Phe, Val, Ile), the comparative helix-forming tendencies have been measured for non-polar amino acid residues that have unbranched side-chains, with an ethyl, propyl or butyl group, and also for methionine. The substitutions are made in a 17-residue alanine-based peptide. The results show that straight-chain non-polar amino acids have high helix-forming tendencies compared to beta-branched non-polar amino acids. Restriction of side-chain conformations in the helix, with a corresponding reduction in conformational entropy, is the likely explanation. There is a small increase in helix-forming tendency as the side-chain increases in length from ethyl to butyl, which suggests that a helix-stabilizing hydrophobic interaction is being detected.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

Smart peptide libraries are accessible via enzymatic modifications

Summary The minireview summarizes the recent preparation of the following unusually modified combinatorial peptide collections useful for diagnostics and screening in drug finding. Tissue transglutaminase catalyzes cross couplings with transamidation between Gln and Lys peptide chains resulting in libraries with isopeptide bonds. The enzyme is involved in the triggering of autoantigenic B- and T-cell epitopes of coeliac disease. The microbial enzyme EpiD involved in lantibiotic biosynthesis catalyzes oxidative decarboxylation of C-terminal cysteine residues in peptide libraries transforming peptidyl-cysteines to peptide (2-mercaptovinyl)amides. Novel backbone modified peptide libraries are prepared using oxazole and thiazole building blocks carrying amino acid side chains. These amino acids have been found in many biologically active natural products from marine and microbial organisms such as microcin B17. Dityrosine and isodityrosine linked peptide dimer libraries are accessible by oxidative phenol coupling using horseradish peroxidase. Such structural elements are found for example in the polycyclic glycopeptide antibiotics of the vancomycin type. Microstructured layers of linear and cyclic peptide libraries are generated on transducer surfaces for cellular assays, sensor developments and even chiral recognition. Examples include a light-directed and microstructured electrochemical polymerization of phenol labelled peptides.     

Unnatural RNA display libraries

Combinatorial peptide and protein libraries have now been developed to accommodate unnatural amino acids in a genetically encoded format via in vitro nonsense and sense suppression. General translation features and specific regioselective and stereoselective properties of the ribosome endow these libraries with a broad chemical diversity. Alternatively, amino acid residues can be chemically derivatized post-translationally to add preferred functionality to the encoded peptide. All of these efforts are advancing combinatorial peptide and protein libraries for enhanced ligands against biological targets of interest.     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

噬菌体展示肽在血管导向治疗方面的应用

介绍了一种利用噬菌体肽库的新技术－体内噬菌体展示（n vivo phage display)。这项技术是在活的动物体内进行的肽库筛选。将肽库通过静脉注射到动物体内,因为血管分子内皮的异质性,噬菌体可以选择性地导向不同组织,这样就可以筛到与特定组织特异结合的噬菌体展示肽。动物实验表明,前凋亡小肽和细胞毒素与导各肽偶联后 治疗效果。这项技术应用于临床,一定有助于肿瘤等疾病的导向治疗和造影技术的发展。     

Characterization of N-phosphoryl oligopeptide libraries by ESI-MS and HPLC-MS

Summary N-diisopropyloxyphosphoryl oligopeptide libraries were constructed through the transformation from the homo-oligopeptide libraries, which were synthesized with the assistance of phosphorus oxychloride. The molecular mass distributions of N-phosphoryl oligopeptide libraries were monitored by ESI-MS, and the components identification and quantitative analysis were carried out by HPLC-MS.     

Removing the redundancy from randomised gene libraries

Amino acid substitution plays a vital role in both the molecular engineering of proteins and analysis of structure-activity relationships. High-throughput substitution is achieved by codon randomisation, which generates a library of mutants (a randomised gene library) in a single experiment. For full randomisation, key codons are typically replaced with NNN (64 sequences) or NN(G)(CorT) (32 sequences). This obligates cloning of redundant codons alongside those required to encode the 20 amino acids. As the number of randomised codons increases, there is therefore a progressive loss of randomisation efficiency; the number of genes required per protein rises exponentially. The redundant codons cause amino acids to be represented unevenly; for example, methionine is encoded just once within NNN, whilst arginine is encoded six times. Finally, the organisation of the genetic code makes it impossible to encode functional subsets of amino acids (e.g. polar residues only) in a single experiment. Here, we present a novel solution to randomisation where genetic redundancy is eliminated; the number of different genes equals the number of encoded proteins, regardless of codon number. There is no inherent amino acid bias and any required subset of amino acids may be encoded in one experiment. This generic approach should be widely applicable in studies involving randomisation of proteins.     

A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead–one-peptide library synthesis, using a Tentagel S-NH₂ solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Utilization of oriented peptide libraries to identify substrate motifs selected by ATM

The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.     

Docking of combinatorial peptide libraries into a broadly cross-reactive human IgM

A monoclonal IgM cryoglobulin with diverse binding behavior was isolated from a patient (Mez) with Waldenstr?m's macroglobulinemia. It gave very high titers in the binding of combinatorially synthesized libraries of peptides ranging in size from two to eight residues. The crystal structure of Mez Fv revealed that the binding site was divided into two cavities of unequal volumes with dimensions and chemical properties that were compatible with the binding of peptides. Access to this unique combination of structural information and peptide binding data led us to carry out Mez-peptide docking simulations to gain insight into the Mez binding propensities. In the present article, the results for docking of five peptide libraries are combined with discussions of the methods and approximations involved in the docking process. We analyze the origins of peptide binding affinity for Mez IgM in terms of its cross-reactivity and its structural preferences.     

A method for generation of arbitrary peptide libraries using genomic DNA

Random peptide libraries can be constructed either by in vitro synthesis of random peptides, or through translation of DNA sequences from synthetic random oligonucleotides. Here we describe an alternative way of making arbitrary peptide libraries with high diversity that can be used in screening as random peptide libraries. Genomic DNA digested with a frequent-cutting restriction enzyme recognizing four nucleotides will theoretically consist of small DNA pieces with average length of 256 nucleotides, and on average around 10⁷ fragments can be generated from a genome of 3 × 10⁹ bases. A peptide library translated from these fragments will have sufficient diversity for some protein interaction screening experiments. Moreover, the same genome digested with a different four-cutter enzyme or ligated into different reading frames will result in different nonoverlapping libraries. A series of such libraries could be generated with genomic DNAs from different species. In this study, human genomic DNA was digested with four-cutter restriction enzymes DpnII and Tsp509I, respectively, and cloned into yeast expression vector pGADT7 to generate arbitrary peptide libraries. These libraries were used in yeast two-hybrid assays to screen for binding motifs of the PDZ domain containing protein synectin. Our results showed that in addition to various native carboxy-terminal tails, synectin could also bind to many artificial ones, some of which contained a consensus sequence—(S/T)XC-COOH.     

Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings

In the solid phase preparation of synthetic peptide libraries, equimolarity of the resultant peptides in the mixture simplifies the identification of active compounds. Two primary methods for the preparation of combinatorial peptide mixtures are currently used. In the first method, the starting resin is divided into equal aliquots, individual amino acids are coupled to each aliquot, and the resin is then recombined. This process is repeated for each position. However, due to the physical process, each resin bead contains only one peptide sequence. Statistically, for mixtures of longer sequences, an ever-increasing amount of resin is necessary to ensure complete representation of each peptide in the library. Thus, each peptide will be represented in the library if a sufficient number of resin beads are used. In addition, the concentration of each peptide in the library depends on both the number of mixture positions in the library and the amount of resin used. In the second method, mixtures of amino acids are coupled simultaneously at each addition step. The proportion of each amino acid in the reaction mixture is varied inversely to its reaction rate such that, ideally, an equimolar mixture of each peptide is synthesized. An advantage of this method over the previous method is that each peptide is ensured to be represented in the library, although not necessarily in equimolar amounts. It is known that not only do the coupling rates of each amino acid vary, but the coupling rates of individual amino acids also change when coupled to different amino acid resins. Consequently, in order to obtain equimolar peptide mixtures through the use of mixtures of protected amino acids, the ratio of reaction rates of one amino acid over another must be constant irrespective of the resin-bound amino acid. If this premise is true, this method of synthesis offers a significant advantage over the previous method since, theoretically, equimolar peptide libraries could be synthesized. The influence of the resin-bound amino acid on the relative reaction rates of incoming amino acids was investigated in the current study. © 1994 John Wiley & Sons, Inc.     

Identification of peptide inhibitors of Pseudomonas aeruginosa exotoxin A function using a yeast two-hybrid approach

The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin. A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A. From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain. The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein. In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity. These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.     

Effects of the sequence and size of non-polar residues on self-assembly of amphiphilic peptides

Peptides with alternating hydrophobic and polar amino acids have been shown to form stable beta-sheet secondary structures and self-assemble into hydrogel-like matrices in the presence of physiological salt concentrations. We hypothesized that the sequence and steric size differences of non-polar residues can affect the balance of peptide intermolecular forces in solution that drive self-assembly. To test this hypothesis, we designed a library of artificial amphiphilic peptides based on the sequence (FEFEFKFK)2 by substituting combinations of the non-polar residues glycine, alanine, valine, leucine and isoleucine for phenylalanine. Peptide structure and self-assembly were characterized using scanning electron microscopy, the Thioflavin T assay, transmission electron microscopy, X-ray fiber diffraction and circular dichroism spectroscopy. The sequence and steric size of non-polar residues are shown to cause variations in peptide secondary structures and create significant differences in the matrix morphology of self-assembled peptides.     

Definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase B/matrix metalloproteinase-9 (MMP-9) and TNF-α converting enzyme (TACE/ADAM-17)

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a regulatory and effector metalloproteinase in inflammation. TNF-α is an important proinflammatory cytokine and is released by the action of a Zn²⁺-containing converting enzyme (TACE/ADAM-17). Both metallo-enzymes play important roles during the development of shock syndromes. Combinatorial chemical synthesis and subsequent library deconvolution were previously used to define a peptide inhibitor (Regasepin1) acting, almost to the same degree, on neutrophil collagenase/MMP-8 and MMP-9 in vitro, and protecting mice against lethal endotoxinemia in vivo. We have now extended this approach by incorporating D-form amino acids and residues preferred by TACE. A new peptide library was designed and synthesized, and by deconvolution new peptide inhibitors were defined. These included a TACE-specific inhibitor, an MMP-9- specific inhibitor, and inhibitors for both enzymes.