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Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU)) 1 . How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8 + T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region–complementary-determining region 3β (BV9–CDR3β) motif 2 – 4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide–MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9–CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease. A study shows that cross-reactivity of microbial antigens and self-antigens presented by HLA-B*27 may be important in the pathogenesis of diseases associated with HLA-B*27 and identifies the shared binding motif responsible.

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l −1 . This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications. Non-ribosomal peptide synthetases have now been modified and de novo non-ribosomal peptide synthetases constructed using new assembly points within condensation domains. This approach enabled the production of new-to-nature peptides, including some carrying synthetic amino acids, as well as the generation of peptide libraries.

High-diversity genetically-encoded combinatorial libraries (10 8 −10 13 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 10 6 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 10 8 members—a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 10 6 –10 8 . These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3–19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact. Synthetic peptide libraries can access broad chemical space, but generally examine only ~ 10 6  compounds. Here, the authors show that in-solution affinity selection, interfaced with nLC-MS/MS sequencing, can identify binders from fully randomized synthetic libraries of 10 8 members.

Protein aggregates are associated with numerous diseases. Here we report a platform for the rapid phenotypic selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in Escherichia coli based on phage-assisted continuous evolution (PACE). We developed a new PACE-compatible selection for protein aggregation inhibition and used it to identify cyclic peptides that suppress amyloid-β42 and human islet amyloid polypeptide aggregation. Additionally, we integrated a negative selection that removes false positives and off-target hits, greatly improving cyclic peptide selectivity. We show that selected inhibitors are active when chemically resynthesized in in vitro assays. Our platform provides a powerful approach for the rapid discovery of cyclic peptide inhibitors of protein aggregation and may serve as the basis for the future evolution of cyclic peptides with a broad spectrum of inhibitory activities. A platform for the continuous selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in Escherichia coli was developed. This platform was used to discover cyclic peptides that suppress aggregation of amyloid-β42 and human islet amyloid polypeptide.

The binding specificities of an individual’s antibody repertoire contain a wealth of biological information. They harbor evidence of environmental exposures, allergies, ongoing or emerging autoimmune disease processes, and responses to immunomodulatory therapies, for example. Highly multiplexed methods to comprehensively interrogate antibody-binding specificities have therefore emerged in recent years as important molecular tools. Here, we provide a detailed protocol for performing ‘phage immunoprecipitation sequencing’ (PhIP-Seq), which is a powerful method for analyzing antibody-repertoire binding specificities with high throughput and at low cost. The methodology uses oligonucleotide library synthesis (OLS) to encode proteomic-scale peptide libraries for display on bacteriophage. These libraries are then immunoprecipitated, using an individual’s antibodies, for subsequent analysis by high-throughput DNA sequencing. We have used PhIP-Seq to identify novel self-antigens associated with autoimmune disease, to characterize the self-reactivity of broadly neutralizing HIV antibodies, and in a large international cross-sectional study of exposure to hundreds of human viruses. Compared with alternative array-based techniques, PhIP-Seq is far more scalable in terms of sample throughput and cost per analysis. Cloning and expression of recombinant proteins are not required (versus protein microarrays), and peptide lengths are limited only by DNA synthesis chemistry (up to 90-aa (amino acid) peptides versus the typical 8- to 12-aa length limit of synthetic peptide arrays). Compared with protein microarrays, however, PhIP-Seq libraries lack discontinuous epitopes and post-translational modifications. To increase the accessibility of PhIP-Seq, we provide detailed instructions for the design of phage-displayed peptidome libraries, their immunoprecipitation using serum antibodies, deep sequencing–based measurement of peptide abundances, and statistical determination of peptide enrichments that reflect antibody–peptide interactions. Once a library has been constructed, PhIP-Seq data can be obtained for analysis within a week.

Antigen discovery technologies have largely focused on major histocompatibility complex (MHC) class I-restricted human T cell receptors (TCRs), leaving methods for MHC class II-restricted and mouse TCR reactivities relatively undeveloped. Here we present TCR mapping of antigenic peptides (TCR-MAP), an antigen discovery method that uses a synthetic TCR-stimulated circuit in immortalized T cells to activate sortase-mediated tagging of engineered antigen-presenting cells (APCs) expressing processed peptides on MHCs. Live, tagged APCs can be directly purified for deconvolution by sequencing, enabling TCRs with unknown specificity to be queried against barcoded peptide libraries in a pooled screening context. TCR-MAP accurately captures self-reactivities or viral reactivities with high throughput and sensitivity for both MHC class I-restricted and class II-restricted TCRs. We elucidate problematic cross-reactivities of clinical TCRs targeting the cancer/testis melanoma-associated antigen A3 and discover targets of myocarditis-inciting autoreactive T cells in mice. TCR-MAP has the potential to accelerate T cell antigen discovery efforts in the context of cancer, infectious disease and autoimmunity. T cell-specific antigens are discovered at high throughput without killing target cells.

Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery. Multicyclic peptides have the potential to mimic the antigen-binding potency of the complementarity-determining regions of antibodies, enabling the development of potent peptide binders to challenging targets, but the scaffolds of multicyclic peptides are difficult to engineer. Here, the authors design a range of triscysteine motifs with disulfide-directing capability for regulating the oxidative folding of multicyclic peptides and employ them in the design of disulfide-directed multicyclic peptide libraries for discovery of potent peptide binders.

Next-generation sequencing (NGS) has raised a growing interest in phage display research. Sequencing depth is a pivotal parameter for using NGS. In the current study, we made a side-by-side comparison of two NGS platforms with different sequencing depths, denoted as lower-throughput (LTP) and higher-throughput (HTP). The capacity of these platforms for characterization of the composition, quality, and diversity of the unselected Ph.D.TM-12 Phage Display Peptide Library was investigated. Our results indicated that HTP sequencing detects a considerably higher number of unique sequences compared to the LTP platform, thus covering a broader diversity of the library. We found a larger percentage of singletons, a smaller percentage of repeated sequences, and a greater percentage of distinct sequences in the LTP datasets. These parameters suggest a higher library quality, resulting in potentially misleading information when using LTP sequencing for such assessment. Our observations showed that HTP reveals a broader distribution of peptide frequencies, thus revealing increased heterogeneity of the library by the HTP approach and offering a comparatively higher capacity for distinguishing peptides from each other. Our analyses suggested that LTP and HTP datasets show discrepancies in their peptide composition and position-specific distribution of amino acids within the library. Taken together, these findings lead us to the conclusion that a higher sequencing depth can yield more in-depth insights into the composition of the library and provide a more complete picture of the quality and diversity of phage display peptide libraries.

Macrocyclic peptides represent an attractive drug modality due to their favourable properties and amenability to in vitro evolution techniques such as phage or mRNA display. Although very powerful, these technologies are not without limitations. In this work, we address some of their drawbacks by developing a yeast display-based strategy to generate, screen and characterise structurally diverse disulfide-cyclised peptides. The use of quantitative flow cytometry enables real-time monitoring of the screening of millions of individual macrocyclic peptides, leading to the identification of ligands with good binding properties to five different protein targets. X-ray analysis of a selected ligand in complex with its target reveals optimal shape complementarity and extensive surface interaction, explaining its exquisite affinity and selectivity. The yeast display-based approach described here offers a facile, quantitative and cost-effective alternative to rapidly and efficiently discover and characterise genetically encoded macrocyclic peptide ligands with sufficiently good binding properties against therapeutically relevant targets. Yeast surface display technology enables real-time monitoring and effective screening of libraries of millions of disulfide-cyclised peptides against diverse protein targets. Selected ligands are characterised rapidly and quantitatively without the need for chemical synthesis and purification.

Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions. Validating crosslinking-mass spectrometry workflows is hampered by the lack of a ground truth to assess the robustness of the crosslink identifications. Here, the authors present a synthetic library of crosslinked peptides, enabling unambiguous discrimination of correct and incorrect crosslink identifications.