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PubMed1994

Construction and screening of biological peptide libraries.

Significant advances have been made recently in technology to construct and screen peptide libraries using biological systems. Progress has been achieved in increasing the size of libraries, in controlling affinity of the peptides isolated, and in understanding the constraints imposed by the biology of the expression systems employed. New receptor ligands and substrates for peptide-modifying enzymes have been isolated using these powerful techniques.

Amino Acid Sequence↗
PubMed1999

Gas-phase separations of electrosprayed peptide libraries.

High-resolution ion mobility spectrometry has been combined with time-of-flight mass spectrometry for analysis of a combinatorial peptide library that is expected to contain 676 components. In this approach, the components of a mixture of three residue peptides, having the general form (D)Phe-Xxx-Xxx-CONH2 (where Xxx is randomized over 26 residues including 10 naturally occurring amino acids and 16 synthetic forms) were ionized by electrospray ionization. Ion mobility/time-of-flight distributions have been recorded for all ions using a nested drift(flight) time technique. The improvement in resolving power [(t/delta t) = 100-150 for singly charged ions] was illustrated by analysis of a mixture of tryptic digest peptides using high- and low-resolution instruments. The approach allows many components of the library (e.g., structural, sequence, and stereo isomers) that cannot be distinguished by mass spectrometry alone to be resolved. Impurities due to side reactions appear to be minimal, comprising < 10% of the total ion signal. Direct evidence for approximately 60-70% of the expected peptides is found. Variation in ion abundance for different components indicates that there are differences in solution concentrations or ionization efficiencies for the components.

Amino Acid Sequence↗
PubMed1993

Peptide libraries: criteria and trends.

The development of approaches for preparing peptide libraries, containing millions of different amino acid sequences of a specified length, provides an invaluable resource for characterizing the molecular interactions that underlie many biological processes. Such libraries can also be used for identifying novel sequences that have a desired biological activity.

Amino Acid Sequence↗
PubMed1994

Matrix-assisted laser desorption ionization for rapid determination of the sequences of biologically active peptides isolated from support-bound combinatorial peptide libraries.

A termination synthesis approach has been developed to encode each resin bead in support-bound combinatorial peptide libraries with the information needed to establish the sequence of the full-length products also contained on the beads. Matrix-assisted laser desorption ionization mass spectrometry was then used to rapidly read the appropriate sequences. In addition to rapid peptide sequencing, the technique allows direct assessment of the quality of the synthetic library, since deletion peptides, side-reaction products and incomplete-deprotection products are readily observed. An anti-gp120 monoclonal antibody was screened against a hexapeptide library, and eight active peptides were isolated. Six of the eight peptides were shown to possess the exact recognition sequence for the antibody.

Amino Acid Sequence↗
PubMed1998

The synthesis and screening of a combinatorial peptide library for affinity ligands for glycosylated haemoglobin.

This paper reports the synthesis and screening of a combinatorial peptide library for new affinity ligands for glycosylated haemoglobin (HbA1c), which is an important indicator of diabetes control. The new ligands are suitable for large-scale synthesis and overcome the disadvantages of antibodies (unstable and expensive to produce etc.), while remaining as efficient as antibodies in binding to the analyte. The library consisted of 262,144 hexapeptides synthesised using the one-bead-one-compound technique. The hexapeptides attached onto beads were screened with glycosylated haemoglobin HbA1c. The structures of the peptides exhibiting high affinity were characterised by Edman microsequencing. Computer modelling simulation of one of the lead sequences has shown that this class of ligand has a high affinity and specificity for glycosylated haemoglobin.

Animals↗
PubMed2005

Selecting peptides for use in nanoscale materials using phage-displayed combinatorial peptide libraries.

Materials that combine inorganic components and biological molecules provide a new paradigm for synthesizing nanoscale and larger structures with tailored physical properties. These synthesis techniques utilize the molecular recognition properties of many biological molecules to nucleate and control growth of the nanoscale structure. Phage-displayed peptide libraries are a powerful tool to identify peptides that selectively recognize and bind to a variety of inorganic surfaces that are utilized in electronic and photonic devices. These libraries have been used extensively to study the peptide-mediated nucleation and growth of some metallic and semiconducting materials, and the application to designed nanostructures has been demonstrated.

Bacteriophage M13↗
PubMed1993

Epitope mapping using bacteriophage peptide libraries.

Vast libraries of random peptides displayed on the surface of bacteriophage potentially allow identification of specific ligands for any peptide receptor of interest. The sequence specificity of antibody-peptide interactions allows these libraries to be used to define and localize continuous epitopes. Remarkably clear-cut results have been obtained with some antibodies; however, the technique has not proved to be generally applicable.

Amino Acid Sequence↗
PubMed1997

[Use of a phage peptide library in mapping the group-specific hemagglutinating domain of alpha-virus glycoprotein E2].

Phage display peptide library f88-4/15 (G. P. Smith, USA) was used for mapping the hemagglutination activity domain of glycoprotein E2 of alphaviruses. Using affinity selection and ELISA, we selected the clones binding monoclonal antibody 4H5 to Venezuelan equine encephalomyelitis virus and inhibiting alphavirus hemagglutinating activity. Analysis of the similarity between the peptides amino acid sequences with the alphavirus glycoprotein E2 sequences revealed a structural motive of 4 amino acid residues (HTSR) which was identified in the 85-88 region. Bacteriophages F36 and F19 contained motives corresponding to 102-SXXM-105 and 109-AXXP-112 regions in alphavirus proteins E2. These data permit us to propose that the detected regions are fragments of a group-specific alphavirus hemagglutination domain.

Amino Acid Sequence↗
PubMed2006

Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries.

A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.

Amino Acid Motifs↗
PubMedDate not supplied

Analysis of the CD2 and spliceosomal Sm B/B' polyproline-arginine motifs defined by a monoclonal antibody using a phage-displayed random peptide library.

The cytoplasmic region of the CD2 receptor of lymphocytes contains proline-rich motifs, which are involved in T cell activation and interleukin-2 production. An intracellular CD2 binding protein, CD2BP2, interacts with two tandem PPPPGHR segments of the CD2 tail. CD2BP2 contains a GYF (glycine-tyrosine-phenylalanine) domain that confers binding to these proline-rich sequences. Monoclonal antibody 3E10 that was previously raised against a peptide containing the CD2 PPPPGHR segment reacts with the native CD2 molecule and spliceosomal Sm B/B' proteins. To identify the exact epitope on the CD2 peptide recognized by 3E10, a phage-displayed combinatorial peptide library was used. Analysis of the selected clones revealed that the mAb 3E10 binds preferentially to the motif PxxPPGxR. Experiments using amino acid substitutions with synthetic peptides confirmed the reactivity of mAb 3E10 with this motif. In addition, we show that several similarities exist between this motif and the CD2BP2-GFY recognition motif PPGxR/K. Binding of antibody 3E10 indicates some degree of degeneracy, which is consistent with its ability to recognize structurally related polyproline-arginine motifs found in intracellular proteins including Sm B/B' proteins and other RNA binding proteins. Thus, mAb 3E10 can be used to specifically identify a sub-class of proline-rich motifs, and as such can be used to study the potential role of these proline-rich sequences in mediating protein-protein interactions.

Amino Acid Motifs↗
PubMed1992

Preparation of soluble peptide libraries: application to studies of platelet adhesion sequences.

We describe chemical synthesis of peptide mixtures that equally express many sequence combinations. Using 65 couplings of single amino acids, five mixtures were prepared with the sequences Tyr-Gly-Arg-Gly-Yyy-Xxx-Xxx, where Yyy is Ser, Asp, Arg, Asn, or Glu, and Xxx is any amino acids. Compositional and sequence analyses supported full representation of all amino acids, except isoleucine was deficient in the sixth position. The data suggest formation of a repertoire of 1,900 sequence combinations (5 x 19 x 20). The mixture with Asp as the fifth residue inhibited platelet adhesion to fibronectin more effectively than the other mixtures. Peptide libraries offer a new tool for investigating bioactive peptides.

Amino Acid Sequence↗
PubMed1998

Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose-bound peptide libraries.

The phosphoenolpyruvate(P-pyruvate)-dependent sugar phosphotransferase system (PTS) is a transport and signal-transduction system which is almost ubiquitous in bacteria but does not occur in eucaryotes. It catalyzes the uptake and phosphorylation of carbohydrates and is involved in signal transduction, e.g. catabolite repression, chemotaxis, and allosteric regulation of metabolic enzymes and transporters. EI (Enzyme I of the PTS) is the first and central component of the divergent PTS (P-pyruvate-dependent sugar phosphotransferase system) phosphorylation cascade. Using immobilized combinatorial peptide libraries and phosphorimaging, heptapeptides and octapeptides were identified which selectively inhibit EI in vitro. The IC50 of the best peptides is 30 microM which is close to the K(M) (6 microM) of EI for its natural substrate HPr (histidine containing phosphoryl carrier protein of the PTS). The affinity-selected peptides are better inhibitors than a peptide with the active-site sequence of HPr. The selected peptides contain several basic residues and one aromatic residue which do not occur in the active site of HPr. The large proportion of basic residues most likely reflects charge complementarity to the strongly acidic active-site pocket of EI. Guanidino groups might facilitate by complexation of the phosphoryl group the slow phosphorylation of the peptide.

Amino Acid Sequence↗
PubMed2000

Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies.

Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses. These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors. In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26-10 and 45-20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin. Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs. Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding. They, therefore, appear to bind at the mAbs digoxin-binding sites. These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin.

Amino Acid Sequence↗
PubMed2002

Epitope mapping of antibodies against prostate-specific antigen with use of peptide libraries.

BACKGROUND: Prostate-specific antigen (PSA) is the most important marker for prostate cancer, but PSA concentrations determined by various assays can differ significantly because of differences in specificity of the antibodies used. To identify epitopes recognized by various monoclonal antibodies (MAbs) to PSA, we have isolated peptides that react with the paratopes of these. METHODS: Six anti-PSA MAbs representing three major epitope groups were screened with five cyclic phage display peptide libraries. After selection, the peptide sequences were determined by sequencing of the relevant part of viral DNA. Binding of the phage peptides to the MAbs was monitored by immunoassay. RESULTS: For each MAb, several paratope-binding peptides with distinct sequence motifs were identified, but only approximately 10% showed similarity with the PSA sequence. Some of these correctly predicted the location of the epitopes. By sequential panning of the library with two closely related MAbs, we identified peptides reacting equally with both MAbs. When analyzed against a large panel of PSA MAbs, the peptides generally showed restricted specificity toward the MAb used for selection, but some peptides bound to several related MAbs. CONCLUSIONS: Most of the cyclic peptides selected with PSA MAbs are specific for the MAb used for selection and do not resemble any sequence on the antigen. Peptides reactive with two MAbs recognizing the same epitope can be obtained by sequential panning. This method can be used to predict the location of some epitopes, but additional methods are needed to confirm the result.

Amino Acid Sequence↗
PubMed1996

Identification of peptides mimicking the antigenicity and immunogenicity of conformational epitopes on Japanese encephalitis virus protein using synthetic peptide libraries.

Monoclonal antibodies (mAbs) N.03 and N.08 that recognize conformational epitopes on the prM protein of Japanese encephalitis virus (JEV) were analyzed to identify their peptide ligands by using a novel approach that combined two different synthetic peptide libraries. Immunoscreening of a library containing 20(5) sequences of pentapeptides revealed that the ligands for N.03 and N.08 had motif sequences, (Y/W/F)GG(I/L/M) and (N/Q)WY(D/E), respectively. To select higher-affinity ligands, we synthesized and screened another type of library with 20 peptide mixtures that were based on the identified motif, where only one amino acid position was defined; and the process was reiterated for the remaining undefined positions. Consequently, the peptides YGGIYMNG and QWYDDR were identified as peptide ligands of N.03 and N.08, respectively. These peptides bound specifically to the antigen-combining sites of the mAbs as confirmed by competitive binding assays. Mouse antisera directed against the peptide YGGIYMNG specifically recognized JEV, while those against QWYDDR did not. These data demonstrated that peptide ligands which reproduce or mimic the immunogenicity as well as the antigenicity of conformational epitopes can be at least partly identified using this approach. This approach may be useful for analyzing conformational epitopes, which are generally difficult to characterize, and might provide a step toward vaccine development when applied to protective mAbs.

Antigens, Viral↗
PubMed2005

Specificity of recombinant antibodies generated from multiple sclerosis cerebrospinal fluid probed with a random peptide library.

We generated recombinant antibodies (rAbs) from over-represented IgG sequences expressed by single plasma cells from multiple sclerosis (MS) cerebrospinal fluid (CSF). Panning of a phage-displayed random peptide library with the rAbs revealed several specific peptide sequences. Inhibition assays confirmed specific binding of the peptides to the antigen-binding site of the antibody. The native IgG of MS CSF from which the recombinant antibody was cloned also recognized these peptides. Our data demonstrate that MS rAb reflects the specificity of IgG in the CSF. Thus, the epitopes/mimotopes identified by MS rAb may provide clues to disease-relevant antigens.

Adult↗
PubMed1998

A novel method for selection of chymotrypsin inhibitors from a phage peptide library.

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

Bacteriophages↗
PubMed2002

Multipin peptide libraries for antibody and receptor epitope screening and characterization.

It has been nearly 15 years since the papers describing the fully systematic epitope mapping approach both for the so-called "continuous" epitopes [Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 3998] and "discontinuous" epitopes [Mol. Immunol. 23 (1986) 709] were published. These seminal papers laid the conceptual foundation for all subsequent developments where a combinatorial approach is applied. Dr. Mario Geysen, the 2000 Kilby Laureate, can certainly lay claim to be the "father of combinatorial chemistry" (http://www.kilby.org/laureates.htm). In this review, I will focus on the aspects of the Multipin technology as they apply to antibody and receptor epitope mapping. Much of what will be presented applies equally well to other applications where peptide libraries (PepSets) and combinatorial approaches are used [Rodda, S.J., 1996. T-cell epitope mapping with synthetic peptides and peripheral blood mononuclear cells. In: Morris, G.E. (Eds.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, Chap. 30, p. 363; Int. J. Pept. Protein Res. 42 (1993) 384; J. Biol. Chem. 271 (1996) 5603]. Factors and techniques that influence the use of the Multipin method for successful epitope mapping will be presented.

Combinatorial Chemistry Techniques↗