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PubMed1994

Electrospray mass spectrometry and tandem mass spectrometry of synthetic multicomponent peptide mixtures: determination of composition and purity.

Electrospray (ES) mass spectrometry and tandem mass spectrometry were used to evaluate the composition and purity of synthetic peptide mixtures ("peptide libraries") consisting of 48 peptides in equimolar amounts. For the structurally similar components of the mixtures little mass discrimination effects were found. The ion intensity distribution of the protonated molecular ions was found to be independent of the concentration. The peak heights in the ES mass spectra reflected the number of isobaric peptides in the mixture. By-products formed by incomplete removal of side chain protecting groups were detected fast and conveniently by tandem mass spectrometry. Daughter ion scans were used to identify common structural features of the peptides and the by-products. Parent ion and constant neutral loss scans allowed the detection of all peptides modified with the same protecting group.

Amino Acid Sequence↗
PubMed2004

Hitting the target with bifunctional phage.

Peptide libraries are rich sources of cell-targeting peptides. In this issue of Chemistry & Biology, Pasqualini and coworkers have developed a chimeric cell-targeting phage system that can easily be modified to delivery of a variety of reagents without need for chemical conjugation.

Animals↗
PubMed2006

Virus-epitope vaccine design: informatic matching the HLA-I polymorphism to the virus genome.

Attempts to develop peptide vaccines, based on a limited number of peptides face two problems: HLA polymorphism and the high mutation rate of viral epitopes. We have developed a new genomic method that ensures maximal coverage and thus maximal applicability of the peptide vaccine. The same method also promises a large number of epitopes per HLA to prevent escape via mutations. Our design can be applied swiftly in order to face rapidly emerging viral diseases. We use a genomic scan of all candidate peptides and join them optimally. For a given virus, we use algorithms computing: peptide cleavage probability, transfer through TAP and MHC binding for a large number of HLA alleles. The resulting peptide libraries are pruned for peptides that are not conserved or are too similar to self peptides. We then use a genetic algorithm to produce an optimal protein composed of peptides from this list properly ordered for cleavage. The selected peptides represent an optimal combination to cover all HLA alleles and all viral proteins. We have applied this method to HCV and found that some HCV proteins (mainly envelope proteins) represent much less peptide than expected. A more detailed analysis of the peptide variability shows a balance between the attempts of the immune system to detect less mutating peptides, and the attempts of viruses to mutate peptides and avoid detection by the immune system. In order to show the applicability of our method, we have further used it on HIV-I, Influenza H3N2 and the Avian Flu Viruses.

Amino Acid Sequence↗
PubMed1999

Biodistribution of filamentous phage-Fab in nude mice.

In vivo panning of peptide libraries in mice has allowed the isolation of peptides which target the vasculature of specific organs. The application of this approach to phage displaying Fab fragments (phage-Fab) could lead to the isolation of antibodies which recognize novel tumor antigens. In this study, we have evaluated the biodistribution of phage-Fab in nude mice. Balb/c nude mice were injected intravenously with 10(9) TU of phage displaying the anti-colon cancer Fab c30.6. Blood samples were collected at nine time points over a period of 72 h and three groups of four mice were sacrificed at 4 min, 24 h and 72 h. Normal tissues (liver, colon, spleen, kidneys, lungs, skeletal muscle) and faeces were collected at these time points and the number of viable phage in each sample was determined. The distribution of phage in tissues was also examined by immunohistochemical analysis of paraffin-embedded tissues. Regression analysis of plasma kinetic data showed that the half-life and the volume of distribution of phage was 3.6 h and 1 ml, respectively. Phage uptake occurred predominantly in lungs, kidneys, spleen and liver. Relatively few phage were distributed to colon and muscle, and phage were eliminated from the circulation by 72 h. Immunohistochemical analysis showed phage to be mainly within the vasculature at 4 min, whereas notable phage extravasation was observed at 24 h and 72 h. In conclusion, this study provides information on the in vivo behavior of phage-Fab which will be useful in the design of in vivo panning strategies. By choosing appropriate time points for tissue collection, it may be possible to isolate novel Fabs against both intra- and extravascular targets.

Animals↗
PubMed2000

Synthesis of a statistically exhaustive fluorescent peptide substrate library for profiling protease specificity.

A statistically exhaustive, 8800 compound tripeptidal amidomethylcoumarin library was synthesized as discreet compounds using solid-phase combinatorial chemistry. A subset of the compounds was purified by HPLC and tested in a high-throughput fluorometric assay against several known serine and cysteine proteases to demonstrate the utility of this library for profiling protease substrate specificity.

Chromatography, High Pressure Liquid↗
PubMed2002

In vivo phage display and vascular heterogeneity: implications for targeted medicine.

The vascular endothelium expresses differential receptors depending on the functional state and tissue localization of its cells. A method to characterize this receptor heterogeneity with phage display random peptide libraries has been developed. Using this technology, several peptide ligands have been isolated that home to tissue-specific endothelial cell receptors following intravenous administration. Such peptide ligands, or antibodies directed against specific vascular receptors, can be used to target therapeutic compounds or imaging agents to endothelial cells in vitro and in vivo. Recent advances in the field include identification of novel endothelial receptors expressed differentially in normal and pathological conditions and the isolation of peptides or antibody ligands to such receptors in in vitro assays, in animal models and in a human patient. These milestones, which extend the 'functional map' of the vasculature, should lead to clinical applications in diseases such as cancer and other conditions that exhibit distinct vascular characteristics.

Endothelium, Vascular↗
PubMed1998

Kinetics and specificity of peptide uptake by the oligopeptide transport system of Lactococcus lactis.

To obtain amino acids for growth, Lactococcus lactis uses a proteolytic system to degrade exogenous proteins such as caseins. The extracellular cell wall-attached proteinase PrtP and the oligopeptide transport system Opp mediate the first two steps in the utilization of caseins. beta-Casein is degraded by PrtP to fragments of 5-30 amino acid residues, and only a limited number of peptides are selected from this pool for uptake via Opp. To study the specificity of Opp and the kinetics of peptide uptake in L. lactis in detail, we used the following strategy: (i) the Opp system was overexpressed; (ii) a 4-fold peptidase mutant was used that is unable to degrade KYGK; (iii) iodinated KYGK was used as the reporter peptide; (iv) libraries of peptides, in which one amino acid position is systematically varied, were used as competitive peptides; and (v) peptides were synthesized on the basis of the beta-casein degradation products, their inhibition of KYGK uptake was determined, and the uptake of these peptides was followed by high-performance liquid chromatography (HPLC). These studies indicate that (i) the Opp system can transport a broad range of peptides from 4 up to at least 18 residues with very little preference for particular side chains and (ii) the kinetics of peptide uptake differ for different substrates tested. Whereas class I peptides such as KYGK exhibit normal Michaelis-Menten kinetics, the level of uptake of the majority of peptides (class II) increases sigmoidally with concentration. Different models for explaining the apparent cooperative effects that are observed for peptide uptake are discussed.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Quantitative determination of single-bead metal content from a peptide combinatorial library.

An electrothermal vaporizer inductively coupled plasma mass spectrometer (ETV-ICPMS) was used to quantitatively screen metals bound to single polystyrene (TentaGel) beads with immobilized oligopeptides. Tests were performed using ETV-ICPMS to screen a series of identical beads as well as a series of combinatorial library beads exposed to a multimetal solution composed of Mg2+, Mn2+, Ni2+, Cu2+, Cd2+, Eu2+, and Pb2+. The residual metal content remaining bound to the beads after acid extractions was also analyzed by solid sampling of the entire bead using oxygen ashing in the ETV. Nine beads (80 mesh, 0.25 mmol g(-1) nominal capacity) containing covalently attached polyaspartic acid (PLAsp; n = 20) showed metal extract concentrations in the range of 4-130 ng mL(-1). After normalizing by bead volume, the precision of capacity measurements in a single bead (7-14%) was primarily dictated by analysis error and contributions from bead diameter measurement with negligible contributions, surprisingly, from variations in site density from bead to bead. A sample combinatorial library of the sequence GXXGXXGXXGXX (X = cysteine, aspartic acid, or glutamic acid and G = glycine) (60 mesh, 0.25 mmol g(-1) nominal capacity) was also used to demonstrate the utility of this method. Metal extract concentrations ranged from 1 to 1300 ng mL(-1) with significant concentration variation between beads, indicating the individual selectivity on each bead. For these larger beads, analysis precision (i.e., capacity precision) was further improved to 3-10% due to the overall increase in bead metal content. Through metal extract determinations, ETV-ICPMS was shown to be a viable nondestructive tool for full metal characterization of "hit" sequences belonging to a combinatorial library.

Binding Sites↗
PubMed2000

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.

Amino Acid Sequence↗
PubMed2001

Specific blocking to improve biopanning in biological samples such as serum and hybridoma supernatants.

Screening phage-displayed peptide libraries (biopanning) is an important technique for acquiring peptide ligands and for mapping peptide epitopes recognized by antibodies (Ab). In biological samples, other materials, not only contaminants but also natural constituents, often interfere with biopanning. Capture methods use anchoring Abs that reduce the need for purification of the intended panning target from a crude sample. This capture method is analogous to sandwich ELISA. However, when the target molecule concentration is low in the initial mixture, the panning of a captured target often yields epitopes that bind to the capture antibody rather than the target of interest. We have developed a methodology that utilizes specific blocking reagents of the capture Ab to extend the sensitivity and applicability of the capture approach to phage panning. A flowchart is presented to enable the worker to begin panning with the simplest approach and then to employ sandwich capture and specific blocking reagents as necessary.

Animals↗
PubMed2005

Decoding protein-protein interactions through combinatorial chemistry: sequence specificity of SHP-1, SHP-2, and SHIP SH2 domains.

A general, combinatorial library method for the rapid identification of high-affinity peptide ligands of protein modular domains is reported. The validity of this method has been demonstrated by determining the sequence specificity of four Src homology 2 (SH2) domains derived from protein tyrosine phosphatase SHP-1 and SHP-2 and inositol phosphatase SHIP. A phosphotyrosyl (pY) peptide library was screened against the SH2 domains, and the beads that carry high-affinity ligands of the SH2 domains were identified and peptides were sequenced by partial Edman degradation and mass spectrometry. The results reveal that the N-terminal SH2 domain of SHP-2 is capable of recognizing four different classes of pY peptides. Binding competition studies suggest that the four classes of pY peptides all bind to the same site on the SH2 domain surface. The C-terminal SH2 domains of SHP-1 and SHP-2 and the SHIP SH2 domain each bind to pY peptides of a single consensus sequence. Database searches using the consensus sequences identified most of the known as well as many potential interacting proteins of SHP-1 and/or SHP-2. Several proteins are found to bind to the SH2 domains of SHP-1 and SHP-2 through a new, nonclassical ITIM motif, (V/I/L)XpY(M/L/F)XP, which corresponds to the class IV peptides selected from the pY library. The combinatorial library method should be generally applicable to other protein domains.

Amino Acid Sequence↗
PubMed2001

Specificity assay of serine proteinases by reverse-phase high-performance liquid chromatography analysis of competing oligopeptide substrate library.

In this paper we present an HPLC method developed for quick activity and specificity analysis of serine proteinases. The method applies a carefully designed peptide library in which the individual components differ only at the potential cleavage site for enzymes. The library has seven members representing seven different cleavage sites and it offers substrates for both trypsin and chymotrypsin-like enzymes. The individual peptide substrates compete for the proteinase during the enzymatic reaction. The reaction is monitored by RP-HPLC separation of the components. We describe the systematic design of the competitive peptide substrate library and the test of the system with eight different serine proteinases. The specificity profiles of the investigated enzymes as determined by the new method were essentially identical to the ones reported in the literature, verifying the ability of the system to characterize substrate specificity. The tests also demonstrated that the system could detect even subtle specificity differences of two isoforms of an enzyme. In addition to recording qualitative specificity profiles, data provided by the system can be analyzed quantitatively, yielding specificity constant values. This method can be a useful tool for quick analysis of uncharacterized gene products as well as new forms of enzymes generated by protein engineering.

Amino Acid Sequence↗
PubMed1997

Identification and characterisation of C1q-binding phage displayed peptides.

Five phage displayed peptide libraries were screened for binders to C1q, the recognition subunit of the classical complement pathway. Two rounds of panning resulted in the isolation and characterisation of several different phage displayed C1q-binding peptides from all five libraries. Two groups of the characterised peptides show sequence similarity with part of the metal ion dependent adhesion site (MIDAS) of integrin A-domains, and the site 187LRNPCPNKEKECQPPF of CD18 (integrin beta2), respectively. These results support binding of complement receptor 3 (CR3, CD11b/CD18, Mac1) to C1q and further suggest C1q binding sites in CR3. We also discuss sequence matches between the characterised peptides and proteins known to interact with C1q, as well as other proteins listed in the SwissProt databank. These findings are of interest for the study of the complement system and may lead to the development of peptides, fusion products or peptido-mimetics with C1q modulating potential.

Amino Acid Sequence↗