Individual Peptides Research Articles

Binding of Natural Antibodies Generated after COVID-19 and Vaccination with Individual Peptides Corresponding to the SARS-CoV-2 S-Protein.

The rapid development of vaccines is a crucial objective in modern biotechnology and molecular pharmacology. In this context, conducting research to expedite the selection of a potent immunogen is imperative. The candidate vaccine should induce the production of antibodies that can recognize the immunogenic epitopes of the target protein, resembling the ones found in recovered patients. One major challenge in vaccine development is the absence of straightforward and reliable techniques to determine the extent to which the spectrum of antibodies produced after vaccination corresponds to antibodies found after recovery. This paper describes a newly developed method to detect antibodies specific to immunogenic epitopes of the target protein in blood plasma and to compare them with antibody spectra generated post vaccination. Comparing the antibody pool generated in the human body after recovering from an infectious disease with the pool formed through vaccination can become a universal method for screening candidate vaccines. This method will enable the identification of candidate vaccines that can induce the production of antibodies similar to those generated in response to a natural infection. Implementing this approach will facilitate the rapid development of new vaccines, even when faced with a pandemic.

Elevated plasma levels of calcitonin gene-related peptide in individuals with rosacea: A cross-sectional case-control study.

Understanding the role of calcitonin gene-related peptide (CGRP) in the pathogenesis of rosacea might provide new therapeutic avenues for individuals with this disease. To compare plasma levels of CGRP between individuals with rosacea and healthy controls. In this cross-sectional case-control study conducted in Copenhagen, Denmark, we collected blood samples from the antecubital vein from adults with rosacea and from healthy controls. We enrolled 123 individuals with rosacea and 68 healthy controls. After adjusting for age and sex, plasma levels of CGRP were significantly higher in individuals with rosacea (mean, 95% confidence interval: 140.21 pmol/L, 128.50-151.92 pmol/L), compared with controls (110.77 pmol/L, 99.91-120.14 pmol/L, p = 0.002). Plasma levels of CGRP were not affected by age, sex, BMI, concomitant migraine, rosacea sub- or phenotype, concomitant disease or current treatment. Participants were not age-, sex- and BMI-matched. Elevated plasma levels of CGRP in individuals with rosacea suggest a role of CGRP in the pathogenesis of rosacea. Targeting CGRP signalling might hold therapeutic promise in people affected by this disease. NCT03872050.

431 Defective uromodulin polymerization and peptide excretion in a natural canine model of kidney stones

OBJECTIVES/GOALS: Using a natural canine model of kidney stone disease, we previously identified a pathogenic variant in the uromodulin gene (UMOD) that imparts a dramatic risk for calcium oxalate (CaOx) stones. This study was designed to characterize the effects of the pathogenic variant on uromodulin processing, specifically polymerization and peptide excretion. METHODS/STUDY POPULATION: Uromodulin polymerization status and peptides were measured in random urine samples from CaOx stone-forming dogs with the pathogenic UMOD variant and breed-, sex-, and age-matched healthy control dogs. Polymerization status was determined using an ultracentrifugation protocol and Western blotting in 6 CaOx cases and 3 controls; relative abundance of the polymerizing and nonpolymerizing forms was evaluated. Uromodulin peptide abundances were measured by LC-MS/MS with 4 dogs per group; results were summed to determine total uromodulin peptide excretion for each dog, and individual peptide abundances were calculated as a percentage of the total. Polymerization status and peptides were compared between groups. RESULTS/ANTICIPATED RESULTS: Dogs with the pathogenic UMOD variant had abnormalities in both uromodulin polymerization and peptide processing. The polymerization data showed that the polymerizing form of uromodulin was abundant in all healthy controls but absent or severely reduced in most dogs with the variant. In contrast, nonpolymerizing uromodulin was detected in all dogs with no observed difference between those with and without the variant. The peptidomics data showed that stone-forming dogs with the pathogenic UMOD variant lacked a peptide cleavage site, resulting in the loss of two common peptides that terminate at that site and the presence of longer peptides that span the site. DISCUSSION/SIGNIFICANCE: These findings implicate uromodulin polymerization and peptide processing defects in kidney stone risk. Future studies will define the mechanisms through which these defects affect stone formation, ultimately informing development of novel preventative therapies.

Toward an Ultimate Solution for Peptide Retention Time Prediction: The Effect of Column Temperature on Separation Selectivity.

We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of ∼100,000 and ∼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of ∼20% of tryptic peptides that were amenable to MS/MS-based identification.

Expression of Multicopy Tandem Recombinant Ginseng Hexapeptide in Bacillus subtilis and the Evaluation of Antiaging Activity.

Ginseng oligopeptides are naturally occurring small-molecule peptides extracted from ginseng that exhibit positive effects on health and longevity. However, the current industrial production of ginseng oligopeptides primarily relies on plant extraction and chemical synthesis. In this study, we proposed a novel genetic engineering approach to produce active ginseng peptides through multicopy tandem insertion (5 and 15 times). The recombinant ginseng peptides were successfully produced from engineered Bacillus subtilis with an increasing yield from 356.55 to 2900 mg/L as the repeats multiple. Additionally, an oxidative stress-induced aging model caused by H2O2 was established to evaluate whether the recombinant ginseng peptides, without enzymatic hydrolysis into individual peptides, also have positive effects on antiaging. The results demonstrated that all two kinds of recombinant ginseng peptides could also delay cellular aging through various mechanisms, such as inhibiting cell cycle arrest, suppressing the expression of pro-inflammatory factors, and enhancing cellular antioxidant capacity.

Abstract 1838: Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si

Abstract Protein sequencing has the potential of playing a crucial role in advancing cancer research by providing insights into the specific proteins involved in cancer pathways. New discoveries from protein sequencing advance our understanding of cancer and potential targeted therapies. Here, we demonstrate the power of next-generation protein sequencing™ using the insight-generating Platinum™ platform. Individual peptides are digested from proteins and immobilized on a semiconductor chip and probed by dye-labeled N-terminal amino acid (NAA) recognizers, followed by subsequent aminopeptidase cleavage to expose each NAA in the peptide for recognition. By recording and analyzing fluorescent intensity, lifetime, and binding kinetics of each NAA binding event, we can decipher single amino acid substitutions and modifications. To demonstrate the versatility of this methodology across proteins associated with cellular biology, we sequenced a series of interleukins: IL4, IL6, IL7, IL13, IL20, and IL34. Interleukins, a group of cytokines that play a vital role in cancer by modulating immune responses, promoting inflammation, and influencing tumor microenvironment, impacting tumor growth and the efficacy of treatments. We have successfully sequenced all the mentioned interleukins individually or within protein mixtures. Furthermore, we were able to sequence IL6 that was enriched through immunoprecipitation from human serum. Finally, we demonstrated the unique potential of Platinum in detecting post-translational modifications (PTMs) relevant to cancer, as exemplified by the successful identification of arginine citrullination in vimentin, an onco-protein that promotes cancer cell mobility and invasion into surround tissues. These results demonstrate the transformative potential of next-generation protein sequencing using Platinum™ in enabling the identification of proteins in complex mixtures and biofluids, as well as the detection of critical PTMs implicated in human pathophysiology, offering valuable insights into cancer research. Citation Format: Khanh Dinh Nguyen. Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1838.

Abstract 3110: An enhanced and cost-efficient method for neoantigen immunogenicity screening method: Double Barcode Neoepitope ScanTM

Abstract Neoantigens, HLA bound peptides generated by tumor-specific mutations, have emerged as promising targets for cancer immunotherapeutic. However, the significant challenge in neoantigen-based therapies is that the neoantigens are rare and difficult to predict. Therefore, in addition to prediction algorithms, the optimization of validation methods is essential for the reliable selection of highly immunogenic neoepitopes. In this study, we have developed a cost-effective screening method to validate large scale of neoantigen candidates. The DNA library-based methods have been employed for antigen selection and T cell receptor identification. We improved the DNA library-based neoantigen screening method by adding two barcodes to each minigene transcribing multiple neoantigen candidates and pooled synthesis of minigenes. We initially designed the barcodes to allow analysis in a 10 by 10 format for screening of hundreds of neoantigens so that PCR with each barcode result in amplification of ten candidates. The second barcode allowed the deconvolution of immunogenicity results without the need for synthesis of individual peptides. In this method B cell was used as antigen-presenting cell, which were co-transfected with mRNA for a minigene pool, 41BBL, and OX40L after expansion with CD40L/IL4/IL21. The resulting B cells were co-cultured with T cells and T cell responses were evaluated using ELISpot. To validate the method the TESLA consortium reported tetramer positive neoantigens were included in minigene. In results, the neoantigens including each barcode were amplified to form a pool of ten neoantigens as a minigene and confirmed that the neoantigens were equally amplified by nested PCR. The neoantigen pools synthesized as mRNA were transfected for presentation in B cells. The result of the targeting a neoantigen pool containing tetramer-positive neoantigens of the TESLA consortium showed a positive for ELISpot. In conclusion, our optimized screening method, employing the double barcode system, offers a time- and cost-efficient approach for the selection of immunogenic neoantigens without the need for individual peptides. This method will facilitate large-scale screening to identify actual neoantigens with immunogenic potential, contributing to the development of neoantigen-targeted vaccines. Citation Format: Seong Gwang Kim, Min-Ho Jung, Hyun-Ji Hwang, Sejin Oh, Seong-Eui Hong, Soonmyung Paik, Hae-Suk Kim. An enhanced and cost-efficient method for neoantigen immunogenicity screening method: Double Barcode Neoepitope ScanTM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3110.

Abstract 1170: An off-the-shelf vaccine activates mutant KRAS-specific T cells in patients with resected pancreatic cancer

Abstract Background: Oncogenic mutations in KRAS (mKRAS) are expressed in up to 90% of pancreatic ductal adenocarcinomas (PDAC) therefore representing a promising immunotherapeutic target. We developed a pooled mKRAS peptide vaccine targeting the 6 most common mutations in PDAC: G12V, G12A, G12C, G12R, G12D, or G13D (NCT04117087). In a phase I study, we evaluated mKRAS-specific T cell responses raised by the vaccine when given in combination with immune checkpoint inhibitors to PDAC patients following resection and adjuvant chemotherapy. Materials and Methods: Patients received the pooled mKRAS peptide vaccine (0.3mg/peptide and 0.5mg poly-ICLC weekly for 4 doses in combination with ipilimumab and nivolumab followed by boosters every 8 weeks with nivolumab. To detect vaccine-induced mKRAS-specific T cells, pre- and post-vaccine peripheral blood mononuclear cells (PBMCs) were restimulated with control or individual mKRAS peptides and IFNγ release was measured by ELISPOT. mKRAS-specific T cell activation, proliferation, memory, and exhaustion profiles were assessed by CyTOF and cytokine secretion was determined by ELISA. For a subset of patients, mKRAS-specific T cells were expanded in vitro and T cell receptor (TCR) β chain sequencing and scRNA/TCRseq were used to identify the KRAS mutation-specific TCR repertoires and define their phenotypes in circulation. Results: mKRAS-specific T cells were detected by IFNγ ELISPOT post-vaccination for all patients, although magnitude and mutation-specific responses varied between patients. CyTOF analysis identified mKRAS-specific Th1 CD4+ central memory (cm, CCR7+IFNγ+) and effector memory (em, CCR7-IFNγ +IL2+ TNFɑ+) responses as well as CD8+ effector (eff, CD137+GZMBhiKi67hi) and effector memory (CCR7-CD137+GZMB+) responses. mKRAS-specific T cells were also detectable in the extended booster phase of treatment (>1 year post initial vaccination). In vitro expanded mKRAS-specific T cells mapped to CD4+ Tcm/em and CD8+ Tem populations within single cell datasets of unstimulated PBMCs collected post-vaccine. 5-25% of the mKRAS-specific T cells significantly expanded to more than one mKRAS antigen suggesting potential cross-reactive T cell clonotypes. In addition, shared public mKRAS-specific T cell clonotypes were identified across patients. Conclusions: This study demonstrates mKRAS vaccine induction of de novo, high quality mKRAS-specific polyfunctional CD4+ and CD8+ T cells. mKRAS-specific TCR analysis identified shared public TCRs across vaccinated patients that have important implications for future “off the shelf” adoptive therapy approaches. Ongoing studies are aimed to validate antigen specificity and map HLA-restriction of mKRAS-specific T cells. Overall, this study demonstrates a positive immunogenic signal of this pooled mutant KRAS vaccine that may be amenable for interception vaccination strategies against PDAC. Citation Format: Amanda L. Huff, Alex Girgis, Saurav D. Haldar, Emily Davis-Marcisak, Thatcher Heumann, Gabriella Longway, Lalitya Andaloori, Alexei Hernandez, Maximilian F. Konig, Brian Mog, Ludmila Danilova, Luciane T. Kagohara, Julie M. Nauroth, Amy M. Thomas, Elana J. Fertig, Won Jin Ho, Elizabeth M. Jaffee, Nilofer Azad, Neeha Zaidi. An off-the-shelf vaccine activates mutant KRAS-specific T cells in patients with resected pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1170.

Abstract 1781: Elucidation of glutathione related dipeptide metabolism in cancer

Abstract There is a long-standing belief that supplementation with antioxidants could potentially treat diseases like cancer. Recent year studies showed, however, that they can behave like a double-edged sword and can also increase tumor proliferation and metastatic progression. The exact mechanism behind antioxidants' impact on tumors is poorly understood. Glutathione (GSH), the most abundant antioxidant in mammalian cells, is a tripeptide containing glutamate, cysteine, and glycine. GSH levels are reported to be significantly elevated within the extracellular space of the tumor. Our preliminary findings show that the breakdown of extracellular GSH can support tumor cell survival under cystine-free conditions. Extracellular GSH catabolism releases cysteinylglycine, which can enter the cell through a dipeptide transporter and be further broken down to supply cysteine and glycine to cells. Alternatively, cysteinylglycine can also be broken down extracellularly into cysteine and glycine, allowing for the uptake of these individual peptides. The molecular mechanisms surrounding the utilization of GSH-breakdown products by the tumor cell are largely unknown. We hypothesize that the proteins responsible for the supply of cysteinylglycine to the tumor cell are required for tumor growth and survival. To test this, we will use unbiased genetic and pharmacologic screening approaches to elucidate the proteins involved in cysteinylglycine processing. Preliminarily, we have found that inhibitors of dipeptidases prevent the ability of cysteinylglycine to support tumor cell survival under cystine starvation, suggesting that tumors require downstream utilization of GSH products. By identifying the mechanisms involved in cysteinylglycine breakdown and supply to tumor cells, we will potentially reveal novel therapeutic targets for cancer treatment, including those not relied upon by normal tissues. Further, our studies will potentially provide a better understanding of how antioxidants impact tumor growth and progression. Citation Format: Fatemeh Alimohammadi, Fabio Hecht, Marco Zocchi, Emily Tuttle, Gloria Asantewaa, TashJae Scales, Nathan Ward, Gina DeNicola, Isaac Harris. Elucidation of glutathione related dipeptide metabolism in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1781.

A set of cross-correlated relaxation experiments to probe the correlation time of two different and complementary spin pairs

Intrinsically disordered proteins (IDPs) defy the conventional structure-function paradigm by lacking a well-defined tertiary structure and exhibiting inherent flexibility. This flexibility leads to distinctive spin relaxation modes, reflecting isolated and specific motions within individual peptide planes. In this work, we propose a new pulse sequence to measure the longitudinal 13C′ CSA-13C′-13Cα DD CCR rate ΓzC′/C′Cα and present a novel 3D version of the transverse ΓxyC′/C′Cα CCR rate, adopting the symmetrical reconversion approach. We combined these rates with the analogous ΓxyN/NH and ΓzN/NH CCR rates to derive residue-specific correlation times for both spin-pairs within the same peptide plane. The presented approach offers a straightforward and intuitive way to compare the correlation times of two different and complementary spin vectors, anticipated to be a valuable aid to determine IDPs backbone dihedral angles distributions. We performed the proposed experiments on two systems: a folded protein ubiquitin and Coturnix japonica osteopontin, a prototypical IDP. Comparative analyses of the results show that the correlation times of different residues vary more for IDPs than globular proteins, indicating that the dynamics of IDPs is largely heterogeneous and dominated by local fluctuations.

Short macrocyclic peptides in sponge genomes

Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.

IDF23-00039 N-terminal pro-brain natriuretic peptide in young aged individuals with cardiometabolic risk factors

IDF23-00039 N-terminal pro-brain natriuretic peptide in young aged individuals with cardiometabolic risk factors

Applications of biaryl cyclization in the synthesis of cyclic enkephalin analogs with a highly restricted flexibility

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta–meta, meta–para, para–meta, and para–para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV β-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.

Development of an Enzyme-Linked Immunosorbent Spot Assay for the Assessment of Adeno-Associated Virus Peptides to Examine Immune Safety.

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)-based and interleukin-2 (IL-2)-based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II-specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2-producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential.

Profiling amyloid-β peptides as biomarkers for cerebral amyloid angiopathy.

Brain amyloid-β (Aβ) deposits are key pathological hallmarks of both cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). Microvascular deposits in CAA mainly consist of the Aβ40 peptide, whereas Aβ42 is the predominant variant in parenchymal plaques in AD. The relevance in pathogenesis and diagnostic accuracy of various other Aβ isoforms in CAA remain understudied. We aimed to investigate the biomarker potential of various Aβ isoforms in cerebrospinal fluid (CSF) to differentiate CAA from AD pathology. We included 25 patients with probable CAA, 50 subjects with a CSF profile indicative of AD pathology (AD-like), and 23 age- and sex-matched controls. CSF levels of Aβ1-34, Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42 were quantified by liquid chromatography mass spectrometry. Lower CSF levels of all six Aβ peptides were observed in CAA patients compared with controls (p = 0.0005-0.03). Except for Aβ1-42 (p = 1.0), all peptides were decreased in CAA compared with AD-like subjects (p = 0.007-0.03). Besides Aβ1-42, none of the Aβ peptides were decreased in AD-like subjects compared with controls. All Aβ peptides combined differentiated CAA from AD-like subjects better (area under the curve [AUC] 0.84) than individual peptide levels (AUC 0.51-0.75). Without Aβ1-42 in the model (since decreased Aβ1-42 served as AD-like selection criterion), the AUC was 0.78 for distinguishing CAA from AD-like subjects. CAA patients and AD-like subjects showed distinct disease-specific CSF Aβ profiles. Peptides shorter than Aβ1-42 were decreased in CAA patients, but not AD-like subjects, which could suggest different pathological mechanisms between vascular and parenchymal Aβ accumulation. This study supports the potential use of this panel of CSF Aβ peptides to indicate presence of CAA pathology with high accuracy.

Methionine Alkylation as an Approach to Quantify Methionine Oxidation Using Mass Spectrometry.

Post-translational oxidation of methionine residues can destabilize proteins or modify their functions. Although levels of methionine oxidation can provide important information regarding the structural integrity and regulation of proteins, their quantitation is often challenging as analytical procedures in and of themselves can artifactually oxidize methionines. Here, we develop a mass-spectrometry-based method called Methionine Oxidation by Blocking with Alkylation (MObBa) that quantifies methionine oxidation by selectively alkylating and blocking unoxidized methionines. Thus, alkylated methionines can be used as a stable proxy for unoxidized methionines. Using proof of concept experiments, we demonstrate that MObBa can be used to measure methionine oxidation levels within individual synthetic peptides and on proteome-wide scales. MObBa may provide a straightforward experimental strategy for mass spectrometric quantitation of methionine oxidation.

MHCpLogics: an interactive machine learning-based tool for unsupervised data visualization and cluster analysis of immunopeptidomes.

The major histocompatibility complex (MHC) encodes a range of immune response genes, including the human leukocyte antigens (HLAs) in humans. These molecules bind peptide antigens and present them on the cell surface for T cell recognition. The repertoires of peptides presented by HLA molecules are termed immunopeptidomes. The highly polymorphic nature of the genres that encode the HLA molecules confers allotype-specific differences in the sequences of bound ligands. Allotype-specific ligand preferences are often defined by peptide-binding motifs. Individuals express up to six classical class I HLA allotypes, which likely present peptides displaying different binding motifs. Such complex datasets make the deconvolution of immunopeptidomic data into allotype-specific contributions and further dissection of binding-specificities challenging. Herein, we developed MHCpLogics as an interactive machine learning-based tool for mining peptide-binding sequence motifs and visualization of immunopeptidome data across complex datasets. We showcase the functionalities of MHCpLogics by analyzing both in-house and published mono- and multi-allelic immunopeptidomics data. The visualization modalities of MHCpLogics allow users to inspect clustered sequences down to individual peptide components and to examine broader sequence patterns within multiple immunopeptidome datasets. MHCpLogics can deconvolute large immunopeptidome datasets enabling the interrogation of clusters for the segregation of allotype-specific peptide sequence motifs, identification of sub-peptidome motifs, and the exportation of clustered peptide sequence lists. The tool facilitates rapid inspection of immunopeptidomes as a resource for the immunology and vaccine communities. MHCpLogics is a standalone application available via an executable installation at: https://github.com/PurcellLab/MHCpLogics.

Abstract B036: Exploration of the human leukocyte antigen immunopeptidome in pancreatic adenocarcinoma

Abstract Pancreatic adenocarcinoma (PDAC) is a lethal disease with an 11% five-year survival rate and frequent distant metastasis at diagnosis. The immune escape mechanisms driving this unchecked proliferation in PDAC remain unclear. The HLA system plays a crucial role in immune-mediated recognition of neoplasms. HLA class I presents endogenously processed peptides to circulating CD8+ T-cells, while HLA class II presents exogenous peptides to CD4+ T-cells, with the collective sum of these peptides known as the immunopeptidome. Most immunopeptidome studies rely on peptides derived solely from cell lines, with few peptides directly reported from patients. Characterization of the immunopeptidome in cancer patients is critical for understanding peptide presentation and neoantigen recognition in PDAC. We hypothesize that isolating HLA class I and II peptides directly from PDAC donors will identify novel neoantigen-derived peptides presented to the immune system with high fidelity for specific HLA alleles. Whole Exome Sequencing (WES) was performed on nine PDAC donors from the UNMC Rapid Autopsy Program. Somatic mutations were identified using best practices published by the BROAD Institute. HLA alleles were identified with the HLA-HD algorithm. Large-scale immunopurification of HLA class I complexes and associated peptides was performed on PDAC liver metastases. Data-Dependent-Acquisition MS/MS with PEAKS analysis identified individual peptide sequences. Similar strategies are being employed for HLA class II peptide isolation from matched donor spleens. To date 39,639 HLA class I peptides and 2,995 HLA class II peptides have been identified from nine donors. Of HLA class I isolated peptides, 9,987 unique sequences have been traced and mapped to the human reference genome. Of the remaining 29,652 unmapped peptides, we have manual confirmation of the presence of neoantigen-derived peptides within that pool, with a pipeline for large scale alignment of these peptides to the annotated mutated genome pending. The gold standard HLA-peptide predictive binding algorithm, netMHC, confirms that 24.2% of our isolated HLA class I peptides are predicted binders for their patient-specific HLA alleles. A notable finding is that 9% of identified HLA class II peptide sequences contained sequences identical to those isolated during independent HLA class I isolations from the same donor. These data establish the high-fidelity of our pipeline for identification of peptides presented by the HLA system directly from PDAC donors. The quantity of HLA-associated peptides discovered sets this study on track to surpass all previously reported patient-derived immunopeptidome studies. The observed conservation of HLA class I and II sequences from independent isolations within the same donor implicates a novel mechanism by which specific combinations of HLA class I and II alleles shape the immunopeptidome. Further work identifying neoantigens in our unmatched isolated HLA class I and II peptides will be continued to uncover potential novel targets for future immunotherapies. Citation Format: William R. Miklavcic, Paul Grandgenett, Michael A. Hollingsworth. Exploration of the human leukocyte antigen immunopeptidome in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B036.

Assessing the immunogenicity risk of salmon calcitonin peptide impurities using in silico and in vitro methods.

Advances in synthetic peptide synthesis have enabled rapid and cost-effective peptide drug manufacturing. For this reason, peptide drugs that were first produced using recombinant DNA (rDNA) technology are now being produced using solid- and liquid-phase peptide synthesis. While peptide synthesis has some advantages over rDNA expression methods, new peptide-related impurities that differ from the active pharmaceutical ingredient (API) may be generated during synthesis. These impurity byproducts of the original peptide sequence feature amino acid insertions, deletions, and side-chain modifications that may alter the immunogenicity risk profile of the drug product. Impurities resulting from synthesis have become the special focus of regulatory review and approval for human use, as outlined in the FDA's Center for Drug Evaluation and Research guidance document, "ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin," published in 2021. This case study illustrates how in silico and in vitro methods can be applied to assess the immunogenicity risk of impurities that may be present in synthetic generic versions of the salmon calcitonin (SCT) drug product. Sponsors of generic drug abbreviated new drug applications (ANDAs) should consider careful control of these impurities (for example, keeping the concentration of the immunogenic impurities below the cut-off recommended by FDA regulators). Twenty example SCT impurities were analyzed using in silico tools and assessed as having slightly more or less immunogenic risk potential relative to the SCT API peptide. Class II human leukocyte antigen (HLA)-binding assays provided independent confirmation that a 9-mer sequence present in the C-terminus of SCT binds promiscuously to multiple HLA DR alleles, while T-cell assays confirmed the expected T-cell responses to SCT and selected impurities. In silico analysis combined with in vitro assays that directly compare the API to each individual impurity peptide may be a useful approach for assessing the potential immunogenic risk posed by peptide impurities that are present in generic drug products.

DRGD-linked charged EKKE dimeric dodecapeptide: pH-based amyloid nanostructures and their application in lead and uranium binding.

Peptides have been reported to undergo self-assembly into diverse nanostructures, influenced by several parameters, including their amino acid sequence, pH, charge, solvent, and temperature. Inspired by natural systems, researchers have developed biomimetic peptides capable of self-assembling into supramolecular functional structures. The present study explored a newly designed peptide sequence, EKKEDRGDEKKE, where E = glutamic acid, K = lysine, D = aspartic acid, G = glycine, and R = arginine, with a metal binding DRGD sequence incorporated between the exclusively charged EKKE peptide. We investigated the formation and the potential of the EKKEDRGDEKKE peptide in retaining the structure and morphology adopted by the individual EKKE peptide. According to a combination of experimental techniques such as thioflavin T fluorescence, field emission-scanning electron microscopy, atomic force microscopy, and circular dichroism, it was evident that the EKKEDRGDEKKE peptide displayed a pH-dependent propensity to adopt amyloid-like structures. Furthermore, the self-assembled entities formed under acidic, basic, and neutral conditions exhibited morphological variations, which resembled that observed for the exclusively charged EKKE peptide. Furthermore, the incorporation of the functional DRGD motif resulted in promising binding to two toxic metal ions, lead (Pb) and uranium (U), as evidenced by a range of spectroscopic techniques, including UV-visible spectroscopy, atomic absorption spectroscopy, fluorescence spectroscopy, and X-ray photoelectron spectroscopy. The use of the amyloid-forming EKKEDRGDEKKE scaffold can also be extended to potential biomedical applications.