Peptide Variants Research Articles

Spritz: A Proteogenomic Database Engine.

Proteoforms are the workhorses of the cell, and subtle differences between their amino acid sequences or post-translational modifications (PTMs) can change their biological function. To most effectively identify and quantify proteoforms in genetically diverse samples by mass spectrometry (MS), it is advantageous to search the MS data against a sample-specific protein database that is tailored to the sample being analyzed, in that it contains the correct amino acid sequences and relevant PTMs for that sample. To this end, we have developed Spritz (https://smith-chem-wisc.github.io/Spritz/), an open-source software tool for generating protein databases annotated with sequence variations and PTMs. We provide a simple graphical user interface for Windows and scripts that can be run on any operating system. Spritz automatically sets up and executes approximately 20 tools, which enable the construction of a proteogenomic database from only raw RNA sequencing data. Sequence variations that are discovered in RNA sequencing data upon comparison to the Ensembl reference genome are annotated on proteins in these databases, and PTM annotations are transferred from UniProt. Modifications can also be discovered and added to the database using bottom-up mass spectrometry data and global PTM discovery in MetaMorpheus. We demonstrate that such sample-specific databases allow the identification of variant peptides, modified variant peptides, and variant proteoforms by searching bottom-up and top-down proteomic data from the Jurkat human T lymphocyte cell line and demonstrate the identification of phosphorylated variant sites with phosphoproteomic data from the U2OS human osteosarcoma cell line.

High Speed AFM and NanoInfrared Spectroscopy Investigation of Aβ1–42 Peptide Variants and Their Interaction With POPC/SM/Chol/GM1 Model Membranes

Due to an aging population, neurodegenerative diseases such as Alzheimer’s disease (AD) have become a major health issue. In the case of AD, Aβ1–42 peptides have been identified as one of the markers of the disease with the formation of senile plaques via their aggregation, and could play a role in memory impairment and other tragic syndromes associated with the disease. Many studies have shown that not only the morphology and structure of Aβ1–42 peptide assembly are playing an important role in the formation of amyloid plaques, but also the interactions between Aβ1–42 and the cellular membrane are crucial regarding the aggregation processes and toxicity of the amyloid peptides. Despite the increasing amount of information on AD associated amyloids and their toxicity, the molecular mechanisms involved still remain unclear and require in-depth investigation at the local scale to clearly decipher the role of the sequence of the amyloid peptides, of their secondary structures, of their oligomeric states, and of their interactions with lipid membranes. In this original study, through the use of Atomic Force Microscopy (AFM) related-techniques, high-speed AFM and nanoInfrared AFM, we tried to unravel at the nanoscale the link between aggregation state, structure and interaction with membranes in the amyloid/membrane interaction. Using three mutants of Aβ peptides, L34T, oG37C, and WT Aβ1–42 peptides, with differences in morphology, structure and assembly process, as well as model lipidic membranes whose composition and structure allow interactions with the peptides, our AFM study coupling high spatial and temporal resolution and nanoscale structure information clearly evidences a local correlation between the secondary structure of the peptides, their fibrillization kinetics and their interactions with model membranes. Membrane disruption is associated to small transient oligomeric entities in the early stages of aggregation that strongly interact with the membrane, and present an antiparallel β-sheet secondary structure. The strong effect on membrane integrity that exists when these oligomeric Aβ1–42 peptides interact with membranes of a particular composition could be a lead for therapeutic studies.

T2238C atrial natriuretic peptide gene variant and cardiovascular events in patients with atrial fibrillation: A substudy from the ATHERO-AF cohort

Background: The T2238C variant of the atrial natriuretic peptide (ANP) gene has emerged as a novel risk factor for the incidence of cardiovascular events. However, the impact of this variant on cardiovascular outcome in patients with atrial fibrillation (AF) is unknown.Methods: We included 557 anticoagulated patients with non-valvular AF randomly selected from the prospective ATHERO-AF cohort. Patients underwent genetic analysis for the T2238C/ANP variant and were grouped as wild type or heterozygous or homozygous for C2238 variant allele. Primary endpoint was a composite of cardiovascular events (CVEs) including cardiovascular death, fatal/non-fatal ischemic stroke and myocardial infarction. Overall, 429 patients carried the TT wild type genotype, 110 patients (19.7%) were heterozygous (T/C) and 18 patients (3.2%) were homozygous (CC).Results: Incidence of CVEs was higher in homozygous patients for C2238 allele at unadjusted analysis (log-rank test, p = 0.042 for additive model, p = 0.043 for recessive model). The multivariable Cox proportional hazards regression analysis confirmed that C2238 ANP allele was associated with CVEs in the additive (p = 0.008) and recessive models (p = 0.005).Conclusions: Carrier status for the C2238/ANP variant allele is associated with an increased risk of CVEs in anticoagulated AF patients.

Heterologous production of new lasso peptide koreensin based on genome mining.

Lasso peptides are a class of ribosomally biosynthesized and posttranslationally modified peptides with a knot structure as a common motif. Based on a genome search, a new biosynthetic gene cluster of lasso peptide was found in the genome of the proteobacterium Sphingomonas koreensis. Interestingly, the amino acid sequence of the precursor peptide gene includes two cell adhesion motif sequences (KGD and DGR). Heterologous production of the new lasso peptide was performed using the cryptic biosynthetic gene cluster of S. koreensis. As a result, a new lasso peptide named koreensin was produced by the gene expression system in the host strain Sphingomonas subterranea. The structure of koreensin was determined by NMR and ESI-MS analysis. The three-dimensional structure of koreensin was obtained based on an NOE experiment and the coupling constants. A variant peptide (koreensin-RGD), which had RGD instead of KGD, was produced by heterologous production with site-directed mutagenesis experiment. Koreensin and koreensin-RGD did not show cell adhesion inhibitory activity, although the molecules possessed cell adhesion motifs. The possible presence of a salt bridge between the motifs in koreensin was indicated, and it may prevent the cell adhesion motif from functioning.

Expression and In Vivo Characterization of the Antimicrobial Peptide Oncocin and Variants Binding to Ribosomes.

Peptides have important biomedical applications, but poor correlation between in vitro and in vivo activities can limit their development for clinical use. The ability to generate peptides and monitor their expression with new mass spectrometric methods and biological activities in vivo would be an advantage for the discovery and improvement of peptide-based drugs. In this study, a plasmid-based system was used to express the ribosome-targeting peptide oncocin (19 amino acids, VDKPPYLPRPRPPRRIYNR) and to determine its direct antibacterial effects on Escherichia coli. Previous biochemical and structure studies showed that oncocin targets the bacterial ribosome. The oncocin peptide generated in vivo strongly inhibits bacterial growth. In vivo dimethyl sulfate footprinting of oncocin on the rRNA gives results that are consistent with those of in vitro studies but reveals additional binding interactions with E. coli ribosomes. Furthermore, expression of truncated or mutated peptides reveals which amino acids are important for antimicrobial activity. Overall, the in vivo peptide expression system can be used to investigate biological activities and interactions of peptides with their targets within the cellular environment and to separate contributions of the sequence to cellular transport. This strategy has future applications for improving the effectiveness of existing peptides and developing new peptide-based drugs.

Abstract 6242: Development of alphalex™-toxin low pH targeting conjugates for the treatment of solid tumors

Abstract Maytansines and their derived maytansinoid DMx compounds are high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Unacceptable dose limiting systemic toxicity has limited the therapeutic potential of these potent anti-oncogenic compounds. Targeting maytansinoids to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. To date Trastuzumab-DM1 (Kadcyla®) remains the only approved antibody-maytansinoid conjugate on the market. Most preclinical maytansinoid conjugates to date face the same issues encountered by Kadcyla® - tumor restriction by target antigen and the potential for off target release of payload. alphalexTM is a tumor targeting technology consisting of a unique variant of pH-Low Insertion Peptide (pHLIP®; references 1-3), cleavable small molecule linker and anti-cancer agent warhead. alphalexTM thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect. Here we demonstrate the ability to conjugate the maytansinoids DM1 and DM4 to alphalexTM via both direct and linker-mediated conjugation. We have demonstrated the ability of our alphalexTM-DM4 conjugate candidates (CBX-13) to have single digit nanomolar potency in vitro as well as exquisitely potent and long-lasting anti-tumor activity in a HER2-negative xenograft model that is un-targetable by competing therapies. In particular we have demonstrated that CBX-13 safely delivers amounts of maytansinoid in vivo that otherwise result in systemic toxicity and death when dosed as free warhead. Based on the SAR of this first generation of maytansinoid conjugates we are further optimizing our alphalexTM - maytansinoid conjugation strategy with the goal of moving forward with IND-enabling studies in the near future.

Abstract 6249: CBX-12: A low pH targeting alphalex™-exatecan conjugate for the treatment of solid tumors

Abstract Topoisomerase inhibitors are potent DNA damaging agents with great potential as anti-cancer drugs for a wide range of solid tumors. However, dose-limiting toxicities such as myelosuppression and gastric toxicity have prevented them from reaching their full clinical potential. Targeting topoisomerase inhibitors with antibodies (i.e. antibody-drug conjugates; ADCs) may enhance the therapeutic window of these agents, but this approach typically limits applicability to a small subset of patients with tumors expressing the target antigen. We recently developed the alphalexTM tumor-targeting platform to overcome the limitations of ADC-based therapeutic strategies. Rather than targeting a specific antigen, alphalexTM consists of a unique variant of pH-Low Insertion Peptide, (pHLIP®; references 1-3) which targets the low pH environment of the tumor, a universal feature characteristic of all tumors due to the Warburg effect. These alphalexTM conjugates form an alpha helix only in low pH conditions, allowing for insertion of the peptide within the cancer cell membrane, delivery of C-terminal warheads across the membrane, and subsequent intracellular release of the agent via glutathione reduction of the linker, thereby allowing for tumor-specific intracellular delivery in an antigen-independent manner. We report the discovery and development of CBX-12, an alphalexTM conjugate of the potent topoisomerase inhibitor, exatecan. CBX-12 provides additional proof of mechanism to the alphalexTM platform by displaying remarkable tumor-targeting properties in preclinical models. CBX-12 displays enhanced stability in plasma in vivo, undergoing only 0.003% warhead release over 30 hours in circulation and demonstrating exquisite selectivity for tumor over normal tissues in mouse tumor models. Notably, CBX-12 allows for efficient delivery of exatecan into tumors due to a highly optimized cleavable linker, allowing CBX-12 to display extraordinary efficacy in a HER2-negative tumor model in an antigen-independent manner. At 10 mg/kg, CBX-12 treatment almost completely suppressed growth of human colorectal tumors in mice, with complete sparing of bone marrow. In contrast, in animals dosed with the equimolar free exatecan (1.15 mg/kg) there was substantial tumor growth accompanied by neutropenia and weight loss. This superior profile of CBX-12 allow us to greatly enhance efficacy relative to dosing equimolar amounts of unconjugated exatecan, which causes significant, dose-limiting bone marrow toxicity. We have demonstrated that CBX-12 is both safe and has potent anti-tumor activity in preclinical models, and we plan to rapidly move forward with the clinical development of CBX-12 as our lead candidate.

Proteomic variations of esophageal squamous cell carcinoma revealed by combining RNA-seq proteogenomics and G-PTM search strategy

BackgroundCancer that arises from epithelial cells of the esophagus is called esophagus squamous cell carcinoma (ESCC) and is mostly observed in developing nations. Evaluation of cancer genomes and its regulation into proteins plays a predominant role in understanding the cancer progressions. Mass-spectrometry-based proteomics is a consequential tool to estimate proteomic variation and posttranslational modifications (PTMs) from standard protein databases. Post-translational modifications play a crucial role in protein folding and PTMs can be accounted for as a biological signal to interpret the structural changes and transition order of proteins. Functional validation of cancer-related mutations can explain the effects of mutations on genes and the identification of Oncogenes and tumor suppressor genes. Therefore, we present a study on protein variations to interpret the structural changes and transition order of proteins in ESCC carcinogenesis. MethodologyWe are using a bottom-up proteomics approach with Galaxy-P framework and RNA sequence data analysis to generate the sample-specific databases containing details of RNA splicing and variant peptides. Once the database generated with information on variable modification, only the curated PTMs at specific positions are considered to perform spectral matching. Proteogenomics mapping was performed to identify protein variations in ESCC. ResultsRNA-sequence proteogenomics with G-PTM (Global Post-Translational Modification) searching strategy has revealed proteomic events including several peptides that contain single amino acid variations, novel splice junction peptides and posttranslationally modified peptides. Proteogenomic mapping exhibited the splice junction peptides mapped predominantly for Malic enzyme exon type (ME-3) and MCM7 protein-coding genes that promote cancer progression, found to be exhibited in ESCC samples. Approximately 25 ± types of PTM modifications were recorded, and Protein Phosphorylation was largely noted. ConclusionESCC cancer prognosis at the molecular level enables a better understanding of cancer carcinogenesis and protein modifications can be used as potential biomarkers.

CusVarDB: A tool for building customized sample-specific variant protein database from next-generation sequencing datasets

Cancer genome sequencing studies have revealed a number of variants in coding regions of several genes. Some of these coding variants play an important role in activating specific pathways that drive proliferation. Coding variants present on cancer cell surfaces by the major histocompatibility complex serve as neo-antigens and result in immune activation. The success of immune therapy in patients is attributed to neo-antigen load on cancer cell surfaces. However, which coding variants are expressed at the protein level can’t be predicted based on genomic data. Complementing genomic data with proteomic data can potentially reveal coding variants that are expressed at the protein level. However, identification of variant peptides using mass spectrometry data is still a challenging task due to the lack of an appropriate tool that integrates genomic and proteomic data analysis pipelines. To overcome this problem, and for the ease of the biologists, we have developed a graphical user interface (GUI)-based tool called CusVarDB. We integrated variant calling pipeline to generate sample-specific variant protein database from next-generation sequencing datasets. We validated the tool with triple negative breast cancer cell line datasets and identified 423, 408, 386 and 361 variant peptides from BT474, MDMAB157, MFM223 and HCC38 datasets, respectively.

Mimicry of a Non-ribosomally Produced Antimicrobial, Brevicidine, by Ribosomal Synthesis and Post-translational Modification

The group of bacterial non-ribosomally produced peptides (NRPs) forms a rich source of antibiotics, such as daptomycin, vancomycin, and teixobactin. The difficulty of functionally expressing and engineering the corresponding large biosynthetic complexes is a bottleneck in developing variants of such peptides. Here, we apply a strategy to synthesize mimics of the recently discovered antimicrobial NRP brevicidine. We mimicked the molecular structure of brevicidine by ribosomally synthesized, post-translationally modified peptide (RiPP) synthesis, introducing several relevant modifications, such as dehydration and thioether ring formation. Following this strategy, in two rounds peptides were engineered invivo, which showed antibacterial activity against Gram-negative pathogenic bacteria susceptible to wild-type brevicidine. This study demonstrates the feasibility of a strategy to structurally and functionally mimic NRPs by employing the synthesis and post-translational modifications typical for RiPPs. This enables the future generation of large genetically encoded peptide libraries of NRP-mimicking structures to screen for antimicrobial activity against relevant pathogens.

Targeted Mass Spectrometry of a Clinically Relevant PSA Variant from Post-DRE Urines for Quantitation and Genotype Determination.

PurposeThe rs17632542 single nucleotide polymorphism (SNP) results in lower serum prostate specific antigen (PSA) levels which may further mitigate against its clinical utility as a prostate cancer biomarker. Post‐digital rectal exam (post‐DRE) urine is a minimally invasive fluid that is currently utilized in prostate cancer diagnosis. To detect and quantitate the variant protein in urine.Experimental designFifty‐three post‐DRE urines from rs17632542 genotyped individuals processed and analyzed by liquid chromatography/mass spectrometry (LC‐MS) in a double‐blinded randomized study. The ability to distinguish between homozygous wild‐type, heterozygous, or homozygous variant is examined before unblinding.ResultsStable‐isotope labeled peptides are used in the detection and quantitation of three peptides of interest in each sample using parallel reaction monitoring (PRM). Using these data, groupings are predicted using hierarchical clustering in R. Accuracy of the predictions show 100% concordance across the 53 samples, including individuals homozygous and heterozygous for the SNP.Conclusions and clinical relevanceThe study demonstrates that MS based peptide variant quantitation in urine could be useful in determining patient genotype expression. This assay provides a tool to evaluate the utility of PSA variant (rs17632542) in parallel with current and forthcoming urine biomarker panels.

An in vitro generated variant of Tephrosia villosa defensin (α-TvD1) enhances biotic stress tolerance in transgenic tobacco

Plant defensins are small, cationic, cysteine-rich peptides with well conserved four disulphide bridges between an α-helix and three anti-parallel β-strands, produced by plants as a first line of innate immunity against the invading fungal pathogens. Some of the plant defensins exhibit strong antibacterial and anti-insect activities in vitro as well as in vivo. TvD1, a defensin from Tephrosia villosa was characterized in our previous studies with demonstrated inhibitory effect against many plant pathogenic fungi and some insects. To further enhance its activity, α-TvD1, a peptide variant of TvD1, was generated through site directed mutagenesis. It showed enhanced antifungal and Tenebrio molitor α-amylase inhibitory activity under in vitro condition. In the present study, we compared the antifungal and anti-insect activities of α-TvD1 with the native TvD1 in transgenic tobacco plants. α-TvD1 transgenic plants showed enhanced tolerance against the oomycetes pathogen Phytophthora parasitica var. nicotianae and the fungal pathogens Alternaria alternata and Rhizoctonia solani. Also, it showed enhanced anti-insect activity against the 1st and 2nd instar larvae of the generalist herbivore, Spodoptera litura. These results demonstrated that α-TvD1 appears to be a potent antimicrobial gene that can be used for generating superior transgenic crops with enhanced resistance against many phytopathogens and insect pests.

Evolution of a splice variant that acts as an endogenous antagonist of the original INSL3 in primates.

Alu sequences are the most abundant repetitive elements in the human genome, and have proliferated to more than one million copies in the human genome. Primate-specific Alu sequences account for ~10% of the human genome, and their spread within the genome has the potential to generate new exons. The new exons produced by Alu elements appear in various primate genes, and their functions have been elucidated. Here, we identified a new exon in the insulin-like 3 gene (INSL3), which evolved ~50 million years ago, and led to a splicing variant with 31 extra amino acid residues in addition to the original 95 nucleotides (NTs) of INSL3. The Alu-INSL3 isoform underwent diverse changes during primate evolution; we identified that human Alu-INSL3 might be on its way to functionality and has potential to antagonize LGR8-INSL3 function. Therefore, the present study is designed to provide an example of the evolutionary trajectory of a variant peptide hormone antagonist that caused by the insertion of an Alu element in primates.

Effect of glutamic acid elimination/substitution on the biological activities of S3 cationic amphiphilic peptides

Cationic amphiphilic peptides (CAPs) are usually classified as bacterial membrane targeting molecules. Rational design and modification of cationic and amphiphilic properties of CAPs have made them to be used in new medical and biotechnological applications. However, CAPs modification and development strategies are challenging issues due to the risk of cytotoxicity or hemolytic activity. In this research, modified variants of S3 peptide were introduced. S3 is a linear 34 amino acid peptide derived from the lipopolysaccharide (LPS) binding site of factor C in horseshoe crab’s hemolymph. Net positive charges of variants (S3E3 and S3E3A) increased by either eliminating negatively charged residues of the peptides or substituting them with alanine. Different biological activities of new variants including LPS binding affinity, antimicrobial activity, cytotoxicity against human breast tumor cell line, and hemolytic property were studied and compared to those of S3 peptide. S3E3 variant showed 68.5% higher LPS binding affinity, 40.4% stronger anti-microbial activity, conserved hemolytic property with the same anti-cancer activity compared to S3peptide. These results revealed that elimination/substitution of negatively charged residues will be a proper strategy for modification of S3 peptide.

4322 Structure-guided design of the TIL1383I T cell receptor

OBJECTIVES/GOALS: Our goal is to employ a structure-guided design approach to engineering a safer and more effective variant of the TIL1383I T cell receptor (TCR) currently under study in clinical trials for malignant melanoma METHODS/STUDY POPULATION: Using our unpublished structure of TIL1383I we are in process of designing a panel of TCR variants with the goal of identifying candidates that improve “focus” towards the tyrosinase antigen presented on the MHC class I molecule HLA-A2. RESULTS/ANTICIPATED RESULTS: Structural analysis of TIL1383I revealed key residues, particularly beta-chain residues E97, G101, L102, responsible for engaging the tyrosinase peptide bound to HLA-A2. The crystal structure of TIL1383I in complex with tyrosinase-HLA-A2 also highlighted its uncharacteristic binding geometry and we therefore hypothesize that this binding orientation is associated with the observed CD8 co-receptor independence of TIL1383I. Indeed, functional analysis with TIL1383I-transduced CD8-positive and CD8-negative T cells, transduced T cells expressing a truncated CD8 lacking the intracellular LCK signaling domain, and tyrosinase peptide variants presented by HLA-A2 mutants outline this co-receptor independence. Combined with our interrogation of tyrosinase peptide cross-reactivity via a peptide positional scanning library approach, structure-guided design resulted in the identification of TIL1383I variants with improved binding affinities to the tyrosinase peptide as well as an understanding of structural characteristics that may contribute to TIL1383I’s co-receptor independence.

Out-of-Register Parallel β-Sheets and Antiparallel β-Sheets Coexist in 150-kDa Oligomers Formed by Amyloid-β(1–42)

We present solid-state NMR measurements of β-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-β peptide (Aβ(1–42)). We build upon our previous report of a β-strand spanned by residues 30–42, which arranges into an antiparallel β-sheet. New results presented here indicate that there is a second β-strand formed by residues 11–24. Contrary to expectations, NMR data indicate that this second β-strand is organized into a parallel β-sheet despite the co-existence of an antiparallel β-sheet in the same structure. In addition, the in-register parallel β-sheet commonly observed for amyloid fibril structure does not apply to residues 11–24 in the 150-kDa oligomer. Rather, we present evidence for an inter-strand registry shift of three residues that likely alternate in direction between adjacent molecules along the β-sheet. We corroborated this unexpected scheme for β-strand organization using multiple two-dimensional NMR and 13C–13C dipolar recoupling experiments. Our findings indicate a previously unknown assembly pathway and inspire a suggestion as to why this aggregate does not grow to larger sizes.

Protease Stability Correlates with Efficacy for High-Affinity, Cell Penetrant MDM2-Binding Stapled Peptides

Nearly two-thirds of all disease-associated proteins are ‘undruggable’ by modern therapeutics, meaning they are inside cells, out of the reach of biologics, but lack small molecule binding pockets. Stabilized peptides have the potential to hit these targets. However, the design criteria for developing agents that can reach and disrupt their target are poorly understood. This work focuses on the physicochemical properties of three newly-developed high affinity double-click stabilized MDM2/p53 inhibiting peptides that have favorable in vitro characteristics including protease stability, cytosolic access, and target-specific cell killing. These properties are distinct fromATSP-7041, the only p53-based peptide that has led to a clinical lead compound. We highlight a promising peptide variant with an added disulfide-bond backbone modification that demonstrates sub-micromolar cell killing in the presence of serum, comparable to the published in vitro potency of ATSP-7041. Overall, these peptides help better define the landscape needed for effective intracellular biologics.

Optimal processing for proteomic genotyping of single human hairs

The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2 cm in length (∼100 μg). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 h of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20 ± 5 to 73 ± 5 (p = 1 × 10−13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.

Novel peptide ligands for antibody purification provide superior clearance of host cell protein impurities

The quest for ligands alternative to Protein A for the purification of monoclonal antibodies (mAbs) has been pursued for almost three decades. Yet, the IgG-binding peptides known to date still fall short of the host cell protein (HCP) logarithmic removal value (LRV) set by Protein A media (2.5-3.1). In this study, we present an integrated computational-experimental approach leading to the discovery of peptide ligands that provide HCP LRVs on par with Protein A. First, the screening of 60,000 peptide variants was performed using a high-throughput search algorithm to identify sequences that ensure IgG affinity binding. Select sequences WQRHGI, MWRGWQ, RHLGWF, and GWLHQR were then negatively screened in silico against a panel of model HCPs to ensure the selection of peptides with high binding selectivity. Candidate ligands WQRHGI and MWRGWQ were conjugated to chromatographic resins and characterized by isothermal binding and breakthrough assays to quantify static and dynamic binding capacity (Qmax and DBC10%), respectively. The resulting Qmax were 52.6 mg of IgG per mL of adsorbent for WQRHGI and 57.48 mg/mL for MWRGWQ, while the DBC10% (2 minutes residence time) were 30.1 mg/mL for WQRHGI and 36.4 mg/mL for MWRGWQ. Evaluation of the peptides by isothermal titration calorimetry (ITC) confirmed the binding energy predicted in silico, and an amino acid scanning study corroborated the affinity-like binding activity of the peptides. WQRHGI-WorkBeads resin was finally characterized by purification of a monoclonal antibody from a Chinese Hamster Ovary (CHO) cell culture harvest, affording a remarkable HCP LRV of 2.7, and consistent product yield and purity over 100 chromatographic cycles. These results demonstrate the potential of WQRHGI as an effective alternative to Protein A for antibody purification.

Natural variants of α-gliadin peptides with wheat proteins with reduced toxicity in coeliac disease - CORRIGENDUM.

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