T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.     

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.     

Thematic minireview series on circular proteins

Circular proteins have now been discovered in all kingdoms of life and are characterized by their exceptional stability and the diversity of their biological activities, primarily in the realm of host defense functions. This thematic minireview series provides an overview of the distribution, evolution, activities, and biological synthesis of circular proteins. It also reviews approaches that biological chemists are taking to develop synthetic methods for making circular proteins in the laboratory. These approaches include solid-phase peptide synthesis based on an adaption of native chemical ligation technology and recombinant DNA approaches that are amenable to the in-cell production of cyclic peptide libraries. The thioester-mediated native chemical ligation approach mimics, to some extent, elements of the natural biosynthetic reaction, which, for disulfide-rich cyclic peptides, appears to involve asparaginyl endopeptidase-mediated processing from larger precursor proteins.     

Generation of accurate peptide retention data for targeted and data independent quantitative LC‐MS analysis: Chromatographic lessons in proteomics

The emergence of data‐independent quantitative LC‐MS/MS analysis protocols further highlights the importance of high‐quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP‐HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP‐HPLC as a “black box”, while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion‐pairing modifier, stationary phase and column temperature, we describe the “mysterious” effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation—a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S‐values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress.     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2

The high level of immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here, we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were suppressed efficiently by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLP-displayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of six, eight and ten amino acid insertions. MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, and they encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity-selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.     

Robust production of a peptide library using methodological synchronization

Peptide libraries have proven to be useful in applications such as substrate profiling, drug candidate screening and identifying protein–protein interaction partners. However, issues of fidelity, peptide length, and purity have been encountered when peptide libraries are chemically synthesized. Biochemically produced libraries, on the other hand, circumvent many of these issues due to the fidelity of the protein synthesis machinery. Using thioredoxin as an expression partner, a stably folded peptide scaffold (avian pancreatic polypeptide) and a compatible cleavage site for human rhinovirus 3C protease, we report a method that allows robust expression of a genetically encoded peptide library, which yields peptides of high purity. In addition, we report the use of methodological synchronization, an experimental design created for the production of a library, from initial cloning to peptide characterization, within a 5-week period of time. Total peptide yields ranged from 0.8% to 16%, which corresponds to 2–70 mg of pure peptide. Additionally, no correlation was observed between the ability to be expressed or overall yield of peptide-fusions and the intrinsic chemical characteristics of the peptides, indicating that this system can be used for a wide variety of peptide sequences with a range of chemical characteristics.     

Extending the coverage of spectral libraries: A neighbor‐based approach to predicting intensities of peptide fragmentation spectra

Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well‐studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor‐based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K‐nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20–60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.     

Peptide and peptidomimetic libraries

Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split-couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.     

Enzyme‐cleavable tandem peptides for quantitative studies in MS‐based proteomics

A novel type of peptide standard is introduced that consists of two peptides combined in one synthetic molecule and separated by a proteolytic cleavage site. Upon enzymatic digestion, the two peptides are released in a molar one‐to‐one ratio. This method enables the generation of exact equimolar mixtures of two peptides of any nature and origin, thereby providing a valuable tool for the investigation of fundamental phenomena in MS. The applicability of the method is exemplified by the analysis of the effect of peptide sequence variations on the relative ionization efficiency in ESI‐ and MALDI‐MS.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

Candidate Biomarker Discovery for Angiogenesis by Automatic Integration of Orbitrap MS1 Spectral-and X!Tandem MS2 Sequencing Information

Candidate protein biomarker discovery by full automatic integration of Orbitrap full MS1 spectral peptide profiling and X!Tandem MS2 peptide sequencing is investigated by analyzing mass spectra from brain tumor samples using Peptrix. Potential protein candidate biomarkers found for angiogenesis are compared with those previously reported in the literature and obtained from previous Fourier transform ion cyclotron resonance (FT-ICR) peptide profiling. Lower mass accuracy of peptide masses measured by Orbitrap compared to those measured by FT-ICR is compensated by the larger number of detected masses separated by liquid chromatography (LC), which can be directly linked to protein identifications. The number of peptide sequences divided by the number of unique sequences is 9248/6911 ≈ 1.3. Peptide sequences appear 1.3 times redundant per up-regulated protein on average in the peptide profile matrix, and do not seem always up-regulated due to tailing in LC retention time (40%), modifications (40%) and mass determination errors (20%). Significantly up-regulated proteins found by integration of X!Tandem are described in the literature as tumor markers and some are linked to angiogenesis. New potential biomarkers are found, but need to be validated independently. Eventually more proteins could be found by actively involving MS2 sequence information in the creation of the MS1 peptide profile matrix.     

Development and validation of a spectral library searching method for peptide identification from MS/MS

A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptides by sequence database searching methods, which often are time-consuming and error-prone. A more precise and efficient method, in which previously observed and identified peptide MS/MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching, is seen as a promising alternative. To that end, an open-source, functionally complete, high-throughput and readily extensible MS/MS spectral searching tool, SpectraST, was developed. A high-quality spectral library was constructed by combining the high-confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines. The resulting library consists of over 30,000 spectra for Saccharomyces cerevisiae. Using this library, SpectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits. A unique advantage of SpectraST is its full integration into the popular Trans Proteomic Pipeline suite of software, which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment, quantification, and data visualization. This method of spectral library searching is especially suited for targeted proteomics applications, offering superior performance to traditional sequence searching.     

ESI-MS Studies of Hetro-peptide Libraries by Phosphorus Oxychloride Activation

Peptide libraries by phosphorus oxychloride activation were developed and several hetro-peptide libraries were synthesized and analyzed by Bio-Tools^TM software with ESI-MS/MS technique. The products of the libraries were studied and the diversity of the peptide libraries was discussed. It was found that the reaction of phosphorus oxychloride with l-Val/l-Leu produced the most abundant hetro-peptide libraries with 68 kinds of peptides sub-libraries based on molecular weight difference.     

α- 促黑激素的固相合成研究

目的:探索α-促黑激素的合成工艺。方法:采用多肽固相合成法制备α-促黑激素。以Rink amide-MBHA树脂为载体、使用Fmoc保护策略、TBTU、HOBt、DIEA为缩合剂体系,最后用TFA、苯甲硫醚、水、苯酚、乙二硫醇混合液将多肽从树脂上切割下来。结果:合成后的目标多肽产率达64.9%,经过RP-HPLC纯化纯度可达98%,质谱鉴定显示纯化产物与目标多肽理论相对分子质量一致。结论:该方法操作方便,反应结果稳定,为固相合成生产α-促黑激素提供了一种可行的工艺方案。     

Solid-phase peptide synthesis in highly loaded conditions

The use of very highly substituted resins has been avoided for peptide synthesis due to the aggravation of chain-chain interactions within beads. To better evaluate this problem, a combined solvation-peptide synthesis approach was herein developed taking as models, several peptide-resins and with peptide contents values increasing up to near 85%. Influence of peptide sequence and loading to solvation characteristics of these compounds was observed. Moreover, chain-chain distance and chain concentration within the bead were also calculated in different loaded conditions. Of note, a severe shrinking of beads occurred during the α-amine deprotonation step only when in heavily loaded resins, thus suggesting the need for the modification of the solvent system at this step. Finally, the yields of different syntheses in low and heavily loaded conditions were comparable, thus indicating the feasibility of applying this latter “prohibitive” chemical synthesis protocol. We thought these results might be basically credited to the possibility, without the need of increasing molar excess of reactants, of carrying out the coupling reaction in higher concentration of reactants - near three to seven folds - favored by the use of smaller amount of resin. Additionally, the alteration in the solvent system at the α-amine deprotonation step might be also improving the peptide synthesis when in heavily loaded experimental protocol.     

Spectrum-to-spectrum searching using a proteome-wide spectral library

The unambiguous assignment of tandem mass spectra (MS/MS) to peptide sequences remains a key unsolved problem in proteomics. Spectral library search strategies have emerged as a promising alternative for peptide identification, in which MS/MS spectra are directly compared against a reference library of confidently assigned spectra. Two problems relate to library size. First, reference spectral libraries are limited to rediscovery of previously identified peptides and are not applicable to new peptides, because of their incomplete coverage of the human proteome. Second, problems arise when searching a spectral library the size of the entire human proteome. We observed that traditional dot product scoring methods do not scale well with spectral library size, showing reduction in sensitivity when library size is increased. We show that this problem can be addressed by optimizing scoring metrics for spectrum-to-spectrum searches with large spectral libraries. MS/MS spectra for the 1.3 million predicted tryptic peptides in the human proteome are simulated using a kinetic fragmentation model (MassAnalyzer version2.1) to create a proteome-wide simulated spectral library. Searches of the simulated library increase MS/MS assignments by 24% compared with Mascot, when using probabilistic and rank based scoring methods. The proteome-wide coverage of the simulated library leads to 11% increase in unique peptide assignments, compared with parallel searches of a reference spectral library. Further improvement is attained when reference spectra and simulated spectra are combined into a hybrid spectral library, yielding 52% increased MS/MS assignments compared with Mascot searches. Our study demonstrates the advantages of using probabilistic and rank based scores to improve performance of spectrum-to-spectrum search strategies.     

An on-bead assay for the identification of non-natural peptides targeting the androgen receptor-cofactor interaction

An efficient and rapid on-bead screening method was established to identify non-natural peptides that target the Androgen Receptor-cofactor interaction. Binding of the Androgen Receptor ligand binding domain to peptide sequences displayed on beads in a One-Bead-One-Compound format could be screened using fluorescence microscopy. The method was applied to generate and screen both a focussed and a random peptide library. Resynthesis of the peptide hits allowed for the verification of the affinity of the selected peptides for the Androgen Receptor in a competitive fluorescence polarization assay. For both libraries strong Androgen Receptor binding peptides were found, both with non-natural and natural amino acids. The peptides identified with natural amino acids showed great similarity in terms of preferred amino acid sequence with peptides previously isolated from biological screens, thus validating the screening approach. The non-natural peptides featured important novel chemical transformations on the relevant hydrophobic amino acid positions interacting with the Androgen Receptor. This screening approach expands the molecular diversity of peptide inhibitors for nuclear receptors.     

Exploiting the human peptidome for novel antimicrobial and anticancer agents

Infectious diseases and cancers are leading causes of death and pose major challenges to public health. The human peptidome encompasses millions of compounds that display an enormous structural and functional diversity and represents an excellent source for the discovery of endogenous agents with antimicrobial and/or anticancer activity. Here, we discuss how to exploit the human peptidome for novel antimicrobial and anticancer agents through the generation of peptide libraries from human body fluids and tissues and stepwise purification of bioactive compounds.     

Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.