Abundant Peptides Research Articles

Improved Chemical and Radiochemical Synthesis of Neuropeptide Y Y2 Receptor Antagonist N-Methyl-JNJ-31020028 and Preclinical Positron Emission Tomography Studies.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals and is involved in several physiological processes through NPY Y1, Y2, Y4 and Y5 receptors. Of those, the Y2 receptor has particular relevance for its autoreceptor role in inhibiting the release of NPY and other neurotransmitters and for its involvement in relevant mechanisms such as feeding behaviour, cognitive processes, emotion regulation, circadian rhythms and disorders such as epilepsy and cancer. PET imaging of the Y2 receptor can provide a valuable platform to understand this receptor's functional role and evaluate its potential as a therapeutic target. In this work, we set out to refine the chemical and radiochemical synthesis of the Y2 receptor antagonist N-[11C]Me-JNJ31020028 for in vivo PET imaging studies. The non-radioactive reference compound, N-Me-JNJ-31020028, was synthesised through batch synthesis and continuous flow methodology, with 43% and 92% yields, respectively. N-[11C]Me-JNJ-31020028 was obtained with a radiochemical purity > 99%, RCY of 31% and molar activity of 156 GBq/μmol. PET imaging clearly showed the tracer's biodistribution in several areas of the mouse brain and gut where Y2 receptors are known to be expressed.

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.

Network analysis of the proteome and peptidome sheds light on human milk as a biological system

Proteins and peptides found in human milk have bioactive potential to benefit the newborn and support healthy development. Research has been carried out on the health benefits of proteins and peptides, but many questions still need to be answered about the nature of these components, how they are formed, and how they end up in the milk. This study explored and elucidated the complexity of the human milk proteome and peptidome. Proteins and peptides were analyzed with non-targeted nanoLC-Orbitrap-MS/MS in a selection of 297 milk samples from the CHILD Cohort Study. Protein and peptide abundances were determined, and a network was inferred using Gaussian graphical modeling (GGM), allowing an investigation of direct associations. This study showed that signatures of (1) specific mechanisms of transport of different groups of proteins, (2) proteolytic degradation by proteases and aminopeptidases, and (3) coagulation and complement activation are present in human milk. These results show the value of an integrated approach in evaluating large-scale omics data sets and provide valuable information for studies that aim to associate protein or peptide profiles from biofluids such as milk with specific physiological characteristics.

Abstract 1244: Exploiting the pathogenesis of endogenous retrovirus to tackle squamous cancers

Abstract Retrotransposons are the evolution relic of ancestrally invaded retroviruses interspersed through our genome. Mostly domesticated as host regulome, retrotransposons shape lineage gene expressions or donate coding functions. A few rare evolutionarily young species remain virally active, leading to germline mutations and modulating immune functions. Aberrant retrotransposon activities have been widely observed in adult diseases, although the extent to which they ensue active viral form and function under those contexts is unclear. Their molecular trigger in adult pathophysiology is poorly defined. To address these questions, we use murine skin as our model, given its well-characterized, abundant and highly accessible adult stem cells, not only mediating postnatal remodeling and homeostatic regeneration, but also responding to stress by driving tissue repair and adaptations. By analyzing a genetic model lacking a known retrotransposon suppressor, histone methyltransferase Setdb1, which we found to be essential in the adult skin, we saw hair loss and hair follicle stem cell exhaustion phenotype, accompanied by a robust and selective surge of endogenous retroviruses (ERVs). When combined with squamous cell carcinoma drivers, Setdb1 loss significantly blocked tumor progression in vivo. Interestingly, compared to relatively mild or no induction of commonly observed evolutionarily young ERVs, the most induced species in our model is a class I ERV, murine leukemia virus (MuLV). We detected abundant retroviral peptides originated from its full-length copies through mass spectrometry and viral-like particles through transmission electron microscopy that are immunoreactive to a monoclonal MuLV env antibody. Similar viral-like particles were broadly evident across several epithelial tissues beyond skin, implicating its conserved function across squamous cancers. Epithelial originated ERVs elicit tissue wide inflammatory response, and can be ameliorated by pharmacological or genetic inhibition of retroviral activity. Physiologically, viral-coding ERVs arise as the epigenetic machinery declines in response to age-associated stress, and plummet when host safeguard prevails in UV induced skin hyperplasia. As an evolutionary conundrum, viral-coding ERVs pose a threat to host fitness and yet, they’ve resisted extinction persisting in the mammalian genome. Our findings suggest viral-coding ERVs wrestle with the host surveillance programs to regulate adult tissue physiology and pathology, thus providing a key target in eliciting innate immunity and tackling squamous cancers. Citation Format: Ying Lyu, Yejing Ge. Exploiting the pathogenesis of endogenous retrovirus to tackle squamous cancers [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 1244.

Abstract 5741: siteDAR, complete antibody drug conjugate, ADC characterization

Abstract Antibody drug conjugate characterization currently extends to the global determination of the drug -to- antibody ratio (DAR). However, there can be confounding batch variability in pharmacological outcome from ADCs of the same drug, antibody, and DAR. Previously, DAR was obtained by quantifying the extent of modification of the intact antibody or its protein subunits. The coupling chemistry is used to infer which type of amino acids were modified but generally no specific sites of modification are identified and if identification is obtained, it is not quantitative. Borrowing the rigorously reproducible proteolytic digestion methods from multi-atribute mapping (MAM) used in monoclonal antibody manufacture, and compiling a comprehensive quantitative assignment of every abundant peptide in the ADC digest we can both calculate a DAR from the ion volume ratios and a quantitative map of the specific modified sites. This method, termed “siteDAR”, is applied here to both commercial Kadcyla® (ado-trastuzumab emtansine) and in-house preparations of ado-trastuzumab emtansine. We show that preparations of Kadcyla® with the same DAR can have significantly different quantitative maps of modification sites. We use siteDAR analysis in our ADC design cycle to characterize an ADC, and have developed an ADC synthesis method that was quantitatively verified, and had increased biosimilarity with commercially available Kadcyla®. siteDAR for commercial Kadcyla siteDAR for initial in-house Kadcyla Initial biosimilarity Citation Format: Warham L. Martin, Michael Senseney, Bhaskar Kusuma, Julien Dugal-Tessier, Nareshkumar Jain. siteDAR, complete antibody drug conjugate, ADC characterization [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 5741.

Abstract 4955: An analytic pipeline of neoantigen selection for both MHC-I and MHC-II pathways

Abstract Background: Accurate neoantigen selection is pivotal for assessing mutation burden and optimizing cancer vaccine formulation thereby playing a crucial role in cancer immunotherapy. The concurrent activation of CD4+ and CD8+ T cells engenders a robust immune response, which is key to effective treatment. However, currently analytic tools predominantly focus on the MHC-I pathway, typically relying on metrics such as gene expression level and MHC-binding affinity of mutated peptides. To address this gap, our study presents a comprehensive pipeline that encompasses both MHC-I and MHC-II pathways and integrates three critical dimensions of immune response: neoantigen abundance, presentation, and immunogenicity. Methods: We estimated mutated peptide abundance by analyzing genomic allelic frequency, transcriptomic allelic frequency, and RNA expression level. Antigen presentation was assessed using the patient harmonic binding rank (PHBR, a composite binding score for all potential MHC-peptide pairs) and MHC diversity (the variety of MHC alleles involved in peptide binding), with additional consideration for the loss of heterozygosity of MHC-I alleles, potentially indicative of immune escape. For immunogenicity, we factored in agretopicity (comparison of MHC-binding affinity between mutated and wild-type peptides), foreignness (similarity to immunogenic foreign antigens), and dissimilarity (sequence disparity with self-peptides, reflecting T cell negative selection process). Results: Implementing our pipeline on a dataset from Parkhurst et al. (2019), we analyzed immune reactivity in relation to 11,537 somatic mutations from 75 gastrointestinal cancer patients. Key metrics—RNA expression, transcriptomic allelic frequencies, PHBR, and MHC diversity—demonstrated significant correlations with both CD4+ and CD8+ activity (p<0.05). Notably, genomic allelic frequency was a significant factor in CD8+ reactivity, whereas agretopicity was crucial for CD4+ reactivity. Conclusion: Our pipeline elucidates the necessity for implementing both MHC-I and MHC-II pathway analyses to effectively select neoantigen candidates for CD8+ and CD4+ T cells respectively. The study confirms that abundance and antigen presentation metrics are robust indicators of neoantigen immunogenicity, supporting their use as potential markers in crafting more effective cancer vaccines. The findings highlight the critical role of this dual-pathway selection approach in generating a comprehensive immune response through neoantigen vaccination. Citation Format: Ko-Han Lee, Timothy Sears, Andrea Castro, Maurizio Zanetti, Hannah Carter. An analytic pipeline of neoantigen selection for both MHC-I and MHC-II pathways [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 4955.

Cultivable microbial diversity, peptide profiles, and bio-functional properties in Parmigiano Reggiano cheese.

Lactic acid bacteria (LAB) communities shape the sensorial and functional properties of artisanal hard-cooked and long-ripened cheeses made with raw bovine milk like Parmigiano Reggiano (PR) cheese. While patterns of microbial evolution have been well studied in PR cheese, there is a lack of information about how this microbial diversity affects the metabolic and functional properties of PR cheese. To fill this information gap, we characterized the cultivable fraction of natural whey starter (NWS) and PR cheeses at different ripening times, both at the species and strain level, and investigated the possible correlation between microbial composition and the evolution of peptide profiles over cheese ripening. The results showed that NWS was a complex community of several biotypes belonging to a few species, namely, Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. lactis. A new species-specific PCR assay was successful in discriminating the cheese-associated species Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lacticaseibacillus zeae. Based on the resolved patterns of species and biotype distribution, Lcb. paracasei and Lcb. zeae were most frequently isolated after 24 and 30 months of ripening, while the number of biotypes was inversely related to the ripening time. Peptidomics analysis revealed more than 520 peptides in cheese samples. To the best of our knowledge, this is the most comprehensive survey of peptides in PR cheese. Most of them were from β-caseins, which represent the best substrate for LAB cell-envelope proteases. The abundance of peptides from β-casein 38-88 region continuously increased during ripening. Remarkably, this region contains precursors for the anti-hypertensive lactotripeptides VPP and IPP, as well as for β-casomorphins. We found that the ripening time strongly affects bioactive peptide profiles and that the occurrence of Lcb. zeae species is positively linked to the incidence of eight anti-hypertensive peptides. This result highlighted how the presence of specific LAB species is likely a pivotal factor in determining PR functional properties.

Use of tryptic peptide MALDI mass spectrometry imaging to identify the spatial proteomic landscape of colorectal cancer liver metastases.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC liver metastases (CRLM) are often resistant to conventional treatments, with high rates of recurrence. Therefore, it is crucial to identify biomarkers for CRLM patients that predict cancer progression. This study utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially map the CRLM tumour proteome. CRLM tissue microarrays (TMAs) of 84 patients were analysed using tryptic peptide MALDI-MSI to spatially monitor peptide abundances across CRLM tissues. Abundance of peptides was compared between tumour vs stroma, male vs female and across three groups of patients based on overall survival (0-3 years, 4-6 years, and 7+ years). Peptides were then characterised and matched using LC-MS/MS. A total of 471 potential peptides were identified by MALDI-MSI. Our results show that two unidentified m/z values (1589.876 and 1092.727) had significantly higher intensities in tumours compared to stroma. Ten m/z values were identified to have correlation with biological sex. Survival analysis identified three peptides (Histone H4, Haemoglobin subunit alpha, and Inosine-5’-monophosphate dehydrogenase 2) and two unidentified m/z values (1305.840 and 1661.060) that were significantly higher in patients with shorter survival (0-3 years relative to 4-6 years and 7+ years). This is the first study using MALDI-MSI, combined with LC-MS/MS, on a large cohort of CRLM patients to identify the spatial proteome in this malignancy. Further, we identify several protein candidates that may be suitable for drug targeting or for future prognostic biomarker development.

Nanoparticle-Catalyzed Transamination under Tumor Microenvironment Conditions: A Novel Tool to Disrupt the Pool of Amino Acids and GSSG in Cancer Cells.

Catalytic cancer therapy targets cancer cells by exploiting the specific characteristics of the tumor microenvironment (TME). TME-based catalytic strategies rely on the use of molecules already present in the TME. Amino groups seem to be a suitable target, given the abundance of proteins and peptides in biological environments. Here we show that catalytic CuFe2O4 nanoparticles are able to foster transaminations with different amino acids and pyruvate, another key molecule present in the TME. We observed a significant in cellulo decrease in glutamine and alanine levels up to 48 h after treatment. In addition, we found that di- and tripeptides also undergo catalytic transamination, thereby extending the range of the effects to other molecules such as glutathione disulfide (GSSG). Mechanistic calculations for GSSG transamination revealed the formation of an imine between the oxo group of pyruvate and the free -NH2 group of GSSG. Our results highlight transamination as alternative to the existing toolbox of catalytic therapies.

Waste to resource: Mining antimicrobial peptides in sludge from metagenomes using machine learning

The emergence of antibiotic-resistant bacteria poses a huge threat to the treatment of infections. Antimicrobial peptides are a class of short peptides that widely exist in organisms and are considered as potential substitutes for traditional antibiotics. Here, we use metagenomics combined with machine learning to find antimicrobial peptides from environmental metagenomes and successfully obtained 16,044,909 predicted AMPs. We compared the abundance of potential antimicrobial peptides in natural environments and engineered environments, and found that engineered environments also have great potential. Further, we chose sludge as a typical engineered environmental sample, and tried to mine antimicrobial peptides from it. Through metaproteome analysis and correlation analysis, we mined 27 candidate AMPs from sludge. We successfully synthesized 25 peptides by chemical synthesis, and experimentally verified that 21 peptides had antibacterial activity against the 4 strains tested. Our work highlights the potential for mining new antimicrobial peptides from engineered environments and demonstrates the effectiveness of mining antimicrobial peptides from sludge.

Structural Characterization and Rat Aortic Vascular Reactivity of Bradykinin-Potentiating Peptides (BPPs) from the Snake Venom of Bothrops moojeni from Delta do Parnaíba Region, Brazil.

Snake venoms contain various bradykinin-potentiating peptides (BPPs). First studied for their vasorelaxant properties due to angiotensin converting enzyme (ACE) inhibition, these molecules present a range of binding partners, among them the argininosuccinate synthase (AsS) enzyme. This has renewed interest in their characterization from biological sources and the evaluation of their pharmacological activities. In the present work, the low molecular weight fraction of Bothrops moojeni venom was obtained and BPPs were characterized by mass spectrometry. Eleven BPPs or related peptides were sequenced, and one of them, BPP-Bm01, was new. Interestingly, some oxidized BPPs were detected. The three most abundant peptides were BPP-Bm01, BPP-Bax12, and BPP-13a, and their putative interactions with the AsS enzyme were investigated in silico. A binding cavity for these molecules was predicted, and docking studies allowed their ranking. Three peptides were synthesized and submitted to vasorelaxation assays using rat aortic rings. While all BPPs were active, BPP-Bm01 showed the highest potency in this assay. This work adds further diversity to BPPs from snake venoms and suggests, for the first time, a putative binding pocket for these molecules in the AsS enzyme. This can guide the design of new and more potent AsS activators.

Dynamics of potential antihypertensive and antidiabetogenic activities during artisanal fermentation of three varieties of Mexican cocoa beans (Theobroma cacao L.)

SummaryThe aim of the present study was to follow the dynamics of potential antihypertensive (angiotensin‐converting enzyme (ACE)) and antidiabetogenic (dipeptidyl peptidase IV (DPP‐IV)) inhibitory activities during artisanal fermentation of Mexican cocoa beans. Almendra Blanca, Criollo and Forastero cocoa beans were sampled after 0, 48, 72, 96 and 120 h of fermentation and roasting (140 °C) and subjected to simulated gastrointestinal digestion. Subsequently, proteolytic activity (OPA method), peptide abundance (RP–HPLC), ACE and DPP‐IV inhibitory activities and IC50 were determined. The results showed that proteolytic activity, peptide abundance and ACE and DPP‐IV inhibition were higher (P < 0.05) at the final stages of fermentation (96 and 120 h). However, IC50 values in partially fermented (48 to 96 h) cocoa beans were the lowest (P < 0.05) of all. In conclusion, a partially fermented cocoa bean may be desirable for its use as a functional ingredient with potential antihypertensive and antidiabetogenic effects.

Pursuing Impactful Quantitative Proteomics Using QC-Channels in Every Spectrum and Trend-Design in Experiment.

False changes discovered by quantitative proteomics reduce the trust of biologists in proteomics and limit the applications of proteomics to unlock biological mechanisms, which suppresses the application of proteomics techniques in the pharmaceutical industry more than it does in academic research. To remove false changes that arise during LC-MS/MS data acquisition, we evaluated the contributions of peptide abundance and number of unique peptides on reproducibility. Lower abundance and only one unique peptide have a higher risk of generating a higher coefficient of variation (CV), resulting in less accurate quantification. However, the abundance of peptides in samples is not adjustable and discarding proteins quantified by only one unique peptide is not a choice either. Indeed, a large percentage of proteins are accurately quantified by only one unique peptide. Therefore, to improve the calculations of the CV, we leverage a new function in PEAKS called QC-channels which enables technical replicates of each spectrum to be evaluated prior to calculation of the CV. While the QC-channels function in PEAKS significantly reduced the false quantification, random false changes still exist due to known or unknown reasons. To address this challenge, we present the idea of Trend-design to track trend changes rather than changes from two points to remove false quantifications and reveal consequential changes responding to a treatment or condition. The idea was confirmed by molecules with different affinity and dose in the current study. The combination of QC-channels and Trend-design enables a more impactful quantitative proteomics to allow unlocking biological mechanisms using proteomics.

Enhancing the techno‐ and bio‐functionality of wheat germ fermented by Lactobacillus plantarum

SummaryThis study explored the fermentation of defatted wheat germ (DWG) using Lactobacillus plantarum, leading to the release of bioactive peptides with improved technological and health properties. Proteomic analysis of fermented defatted wheat germ (FDWG) and DWG revealed a higher abundance of functional peptides in FDWG, with 27 peaks ranging from 503–1391 m/z compared to 22 peaks ranging from 537–1362 m/z for DWG. The peptides identified in FDWG were enriched in hydrophobic amino acids, known for their antioxidant and radical scavenging capabilities. Furthermore, FDWG‐based bread demonstrated the lowest gliadin content, making it a promising option for coeliac patients. The bread also exhibited a soft texture and higher consumer acceptance, positioning it as a viable alternative in reduced‐gluten bakery products. Overall, fermented wheat germ presents an exciting avenue for obtaining bioactive peptides with diverse potential applications in functional foods and nutraceuticals.

Gestational Age-Related Urinary Peptidome Changes in Preterm and Term Born Infants

Introduction: Preterm infants are at risk for a variety of somatic and neurological disorders. In recent years, biofluid proteomics has emerged as a potential diagnostic tool for biomarker analysis. The aim of this study was to determine gestational age (GA)-related patterns of the urinary peptidome in preterm infants for researching potential novel prognostic biomarkers. Methods: We performed urinary peptidomics in longitudinal samples of 24 preterm (mean GA weeks 28 + 1 [24+1–31 + 6]) and 27 term born controls (mean GA weeks 39 + 2 [37+0–41 + 1]) using capillary electrophoresis combined with mass spectrometry (CE-MS). Peptides were sequenced using CE-MS/MS or LC-MS/MS analysis and were deposited, matched, and annotated in a Microsoft SQL database for statistical analysis. We compared their abundance in urine of preterm and term born infants and performed a validation analysis as well as correlations to GA and clinical risk scores. Results: Our results confirmed significant differences in the abundance of peptides and the hypothesis of age-dependent urinary peptidome changes in preterm and term infants. In preterm infants, SLC38A10 (solute carrier family 38 member 10) is one of the most abundant peptides. Combined urinary peptides correlated with clinical risk scores (p < 0.05). Conclusion: This is the first study reporting GA-related urinary peptidome changes of preterm infants detected by CE-MS and a modulation of the peptidome with GA. Further research is required to locate peptidome clusters correlated with specific clinical complications and long-term outcome. This may identify preterm infants at higher risk for adverse outcome who would benefit from early intervention.

Endogenous Production and Vibrational Analysis of Heavy-Isotope-Labeled Peptides from Cyanobacteria.

Stable isotope labeling is an extremely useful tool for characterizing the structure, tracing the metabolism, and imaging the distribution of natural products in living organisms using mass-sensitive measurement techniques. In this study, a cyanobacterium was cultured in 15 N/13 C-enriched media to endogenously produce labeled, bioactive oligopeptides. The extent of heavy isotope incorporation in these peptides was determined with LC-MS, while the overall extent of heavy isotope incorporation in whole cells was studied with nanoSIMS and AFM-IR. Up to 98 % heavy isotope incorporation was observed in labeled cells. Three of the most abundant peptides, microcystin-LR (MCLR), cyanopeptolin-A (CYPA), and aerucyclamide-A (ACAA), were isolated and further studied with Raman and FTIR spectroscopies and DFT calculations. This revealed several IR and Raman active vibrations associated with functional groups not common in ribosomal peptides, like diene, ester, thiazole, thiazoline, and oxazoline groups, which could be suitable for future vibrational imaging studies. More broadly, this study outlines a simple and relatively inexpensive method for producing heavy-labeled natural products. Manipulating the bacterial culture conditions by the addition of specific types and amounts of heavy-labeled nutrients provides an efficient means of producing heavy-labeled natural products for mass-sensitive imaging studies.

Neglected function of gastrin to reduce feeding in Siberian sturgeon (Acipenser baerii) via cholecystokinin receptor B.

Gastrin is an important intragastrointestinal hormone, but reports on its regulation of feeding behavior in fish are still scarce. This study aimed to determine the feeding regulatory function of gastrin in sturgeon. In this study, a gastrin/cholecystokinin-like peptide was identified in the genomes of sturgeon and proved to be gastrin by evolutionary tree analysis. Tissue distribution of gastrin and its receptor, cholecystokinin receptor B (CCKRB), showed that both had high mRNA abundance in the hypothalamus and gastrointestinal tract. In the duodenum, gastrin and CCKRB mRNAs were reduced at 1h of fasting, and both were also observed in the stomach and hypothalamus in response to changes in feeding status. Sulfated gastrin 17 is the major form of gastrin in vivo. Therefore, we investigated the effect of sulfated gastrin 17 on feeding by intraperitoneal injection into Siberian sturgeon using sulfated gastrin 17. The results showed that gastrin 17 significantly reduced the cumulative feeding of Siberian sturgeon in the short term (1, 3 and 6h) and long term (1, 2, 3, 4, 5 and 7days). Finally, we explored the potential mechanism of feeding inhibition after intraperitoneal injection of gastrin 17 for 7 consecutive days. The results showed that gastrin 17 treatment significantly increased the mRNA levels of anorexigenic peptides (cart, cck and pyy), while it had no significant effect on the mRNA abundance of orexigenic peptides (npy and agrp). In addition, gastrin 17 treatment significantly affected the expression of appetite signaling pathways in the hypothalamus, such that the mRNA expression of ampkα1 was significantly reduced, whereas the mRNA abundance of stat3, mtor and s6k was significantly increased. In conclusion, the present study confirmed the anorectic effect of gastrin on Siberian sturgeon.

The Greater Celandine: Identification and Characterization of an Antimicrobial Peptide from Chelidonium majus.

Chelidonium majus, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

IgG glycopeptide enrichment using hydrophilic interaction chromatography-based solid-phase extraction on anaminopropyl column.

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which havenot yet beenconsidered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.

Circular dichroism characterization of late embryogenesis abundant peptides in response to salts, temperature, and reduced hydration

Circular dichroism characterization of late embryogenesis abundant peptides in response to salts, temperature, and reduced hydration