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The aim of this work was to develop, for the first time, sustainable strategies, based on the use of Ultrasound-Assisted Extraction, Natural Deep Eutectic Solvents, and Pressurized Liquid Extraction, to extract proteins from lime (Citrus x latifolia) peels and to evaluate their potential to release bioactive peptides. PLE showed the largest extraction of proteins (66-69%), which were hydrolysed using three different enzymes (Alcalase 2.4 L FG, Alcalase®PURE 2.4 L, and Thermolysin). The in vitro antioxidant and antihypertensive activities of released peptides were evaluated. Although all hydrolysates showed antioxidant and antihypertensive activity, the hydrolysate obtained with Thermolysin showed the most significant values. Since the Total Phenolic Content in all hydrolysates was low, peptides were likely the main contributors to these bioactivities. Hydrolysates were analyzed by UHPLC-QTOF-MS and a total of 98 different peptides were identified. Most of these peptides were rich in amino acids associated with antioxidant activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Adenovirus (AdVs) is the viral vector of choice in vaccines and oncolytic applications owing to its high transduction activity and inherent immunogenicity. For decades, AdV isolation has relied on ultracentrifugation and ion-exchange chromatography, which are not suitable to large-scale production and struggle to deliver sufficient purity. Immunoaffinity chromatography resins of recent introduction feature high binding capacity and selectivity, but mandate harsh elution conditions (pH 3.0), afford low yield (< 20%), and provide limited reusability. Seeking a more efficient and affordable alternative, this study introduces the first peptide affinity ligands for AdV purification. The peptides were identified via combinatorial selection and in silico design to target hexons, the most abundant proteins in the adenoviral capsid. Selected peptide ligands AEFFIWNA and TNDGPDYSSPLTGSG were conjugated on chromatographic resins and utilized to purify AdV serotype 5 from HEK293 and Vero cell lysates. The peptide-functionalized resins feature high binding capacity (> 10 10 active virions per mL at the residence time of 2 min), provide high yield (> 50%) and up to 100-fold reduction of host cell proteins and DNA. Notably, the peptide ligands enable gentle elution conditions (pH 8) that prevent the "shedding" of penton and fiber proteins, thus affording intact adenovirus particles with high cell-transduction activity. The study of the peptide ligands by surface plasmon resonance and molecular docking and dynamics simulations confirmed the selective targeting of hexon proteins and elucidated the molecular-level mechanisms underlying binding and release. Collectively, these results demonstrate the strong promise of peptide ligands presented herein for the affinity purification of AdVs from cell lysates., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, different ultrasound-assisted modes [ultrasonic simultaneous (US) and ultrasonic preconditioning (UP)] of synergistic enzymatic hydrolysis were used to prepare bioactive peptides of sheep hoof collagen. The 2, 2-diphenyl - 1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2 '-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity and metal chelating capacity of sheep's hoof collagen antioxidant peptides (SCPs) (at 1 mg/mL) prepared at 20 min of treatment in US treatment mode (US-20) were 48.56 ± 0.68 %, 51.97 ± 1.15 % and 65.58 ± 1.36 %, respectively, which were higher compared with the control and UP groups. Using LC-MS/MS analysis, 9336, 11,527, and 11,909 peptide sequences were identified from collagen hydrolysate by C, UP-20, and US-20, respectively. The peptides ACEDAPPSAAHFR and FGFEVGPACFLG with high bioactivity were screened using computer analysis. Molecular docking results revealed that hydrogen bonding and hydrophobic interactions between the two peptide sequences with DPPH and ABTS radicals may be responsible for their antioxidant properties. Therefore, we have optimized the extraction of bioactive peptides from sheep hoof collagen using ultrasound-assisted enzymatic hydrolysis, which is helpful for the high-value utilisation of sheep hoof by-products and the extraction of foodborne antioxidant peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Batch coupled enzymatic hydrolysis and membrane separation mode (BCEH-MSM) is efficient in preparing active peptides due to enzyme being more purposeful in hydrolysing macromolecular. Therefore, BCEH-MSM probably could be an alternative option to the traditional enzymatic hydrolysis and offline membrane separation mode (TEH-OMSM). This work aimed to explore the potential of BCEH-MSM in enhancing the enzymatic hydrolysis (EH) efficiency and the umami of the enzymatic hydrolysate. The EH efficiency was valuated based on product yields. Amino acid analyzer and HPLC were used to analyze tasting compounds. Electronic-tongue was used to determine umami intensity. The results showed that BCEH-MSM exhibited superior EH efficiency and higher umami intensity compared to TEH-OMSM. LC-MS/MS was used to identify peptides with higher umami intensity in the enzymatic hydrolysate. LGEETF, VNFDGEI, and QLSELLRAGSSPNL had umami profile verified by electronic-tongue. Molecular docking further showed that crucial amino acid residues involved in the binding to T1R1/T1R3 was His145., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: A sea cucumber ( Stichopus japonicus ) is an invertebrate rich in high-quality protein peptides that inhabits the coastal seas around East Asian countries. Such bioactive peptides can be utilized in targeted disease therapies and practical applications in the nutraceutical industry., Methods: Bioactive peptides were isolated from Stichopus japonicus through ultrafiltration and Sephadex G-10 size exclusion chromatography. The low-molecular-weight fraction (ACSH-III) showed the highest hydroxyl radical scavenging and angiotensin-converting enzyme (ACE) inhibitory activities. Subsequent purification of ACSH-III resulted in four fractions, of which ACSH-III-F3 and ACSH-III-F4 exhibited significant bioactivity., Results: Peptides identified in these fractions, including Phenylalanine-Proline-Threonine-Tyrosine (FPTY) and Tyrosine-Proline-Serine-Tyrosine-Proline-Serine (YPSYPS), were characterized using high-performance liquid chromatography (HPLC) and quadrupole time-of-flight mass spectrometry (QTOF-MS). FPTY demonstrated the most potent antioxidant and antihypertensive activities among these peptides, with IC 50 values of 0.11 ± 0.01 mg/mL for hydroxyl radicals and 0.03 ± 0.01 mg/mL for ACE inhibition. Docking simulations revealed strong binding affinities of these peptides to the active site of the ACE, with FPTY displaying interactions similar to those of the synthetic inhibitor lisinopril., Conclusions: These findings suggest that the identified peptides, particularly FPTY, have potential applications as natural antioxidants and functional foods., (© 2024 The Author(s). Published by IMR Press.)

Whey is a byproduct of the dairy industry and is rich in protein. To enhance the significance of such byproducts and find efficacious antioxidants for combating oxidative stress, this study reported on the preparation, purification, and identification of novel peptides with antioxidant activities from whey protein metabolites following fermentation by Lactobacillus rhamnosus B2-1. The isolation and identification processes involved macroporous adsorption resin column chromatography, gel filtration column chromatography, and liquid chromatography-tandem mass spectrometry. Therein, three novel antioxidant peptides (PKYPVEPF, LEASPEVI, and YPFPGPIHNS) were selected to be synthesized, and they demonstrated remarkable antioxidant activities in vitro chemical assays. PKYPVEPF, LEASPEVI, and YPFPGPIHNS (100 μg/mL) displayed a notable cytoprotective impact on HepG2 cells under oxidative stress induced by H 2 O 2 , increasing the cell viability from 49.02 ± 3.05% to 88.59 ± 10.49%, 82.38 ± 19.16%, and 85.15 ± 7.19%, respectively. Moreover, the peptides boosted the activities of catalase and superoxide dismutase in damaged cells and reduced reactive oxygen species levels. The molecular docking studies highlighted that these antioxidant peptides efficiently bound to key amino acids in the Kelch domain of Keap1, thereby preventing the interaction between Keap1 and Nrf2. In conclusion, PKYPVEPF, LEASPEVI, and YPFPGPIHNS demonstrated substantial antioxidant activity, suggesting their potential for widespread application as functional food additives and ingredients.

Currently, there is a demand for protein sources that do not use soil management or animal breeding. Among these sources we highlight the microorganisms, such cyanobacteria and microalgae, which have a simple growth using light, CO2, water and some mineral salts to generate high protein production. The extraction of these proteins depends on the method used. The most used methods for extracting bio-functional proteins are mechanical, chemical and enzymatic. The aim of this work is to analyze the protein extraction methods in microalgae using Scielo, ScienceDirect and NCBI (PubMed) electronic databases that made it possible to select original studies published in the last five years (2018-2023). A total of 2707 articles, 25 of which were selected for further analysis and subjected to risk of bias assessment. The genera Chlorella, Scenedesmus and Nannochloropsis were the most studied due to their high protein content. Mechanical methods and chemical hydrolysis are the most used methods, achieving an extraction yield of 46.0 % and 64.0 %, respectively. The best extraction results are obtained with a combination of methods, reaching up to 80.0 % yield. However, some aspects need to be observed to choose an ideal protein extraction method.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder associated with significant morbidity, including pruritus, recurrent skin lesions, and immune dysregulation. This study aimed to investigate the antioxidative and anti-AD effects of peptides derived from hydrolyzed Sebastes schlegelii (Korea rockfish) tail by-products. Hydrolysates were prepared using various enzymes, including Alcalase, Flavourzyme, Neutrase, and Protamex. Among them, Protamex hydrolysates demonstrated the highest ABTS radical scavenging activity with an RC 50 value of 69.69 ± 0.41 µg/mL. Peptides were further isolated from the Protamex hydrolysate using dialysis, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). The most active peptide, STPO-B-II, exhibited a single peak and was identified as a sequence of Glu-Leu-Ala-Lys-Thr-Trp-His-Asp-Met-Lys, designated as MP003. In vivo experiments were conducted using a 2,4-dinitrochlorbenzene (DNCB)-induced AD model in NC/Nga mice. The isolated peptide, MP003, showed significantly reduced AD symptoms, including erythema, lichenification, and collagen deposition. Additionally, MP003 decreased epidermal and dermal thickness, eosinophil, and mast cell infiltration and downregulated the expression of pro-inflammatory cytokines IL-1β, IL-6, and IgE in serum and skin tissues. These findings suggest that peptides derived from Sebastes schlegelii tail by-products may serve as potential therapeutic agents for AD.

Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 10 14 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy., Competing Interests: Declaration of competing interest Eduardo Barbieri, Michael Crapanzano, and Stefano Menegatti declare conflict of interest respectively as Senior Scientist, Chief Executive Officer, and Chief Technology Officer of LigaTrap Technologies. The authors Stefano Menegatti and Shriarjun Shastry are the authors of Patent Application PCT/US24/29159, titled "Compositions and Methods for Purifying Viral Vectors", filed on May 13 2024 at 07:56:57 pm (Eastern Time). The application was filed by Peter J. Schlueter at Casimir Jones, S.C. (address: 2275 Deming Way, Suite 310 Middleton, WI 53562, US; email: pjschlueter@casimirjones.com) on behalf of North Carolina State University as the applicant. The invention has been exclusively licensed to LigaTrap Technologies, LLC. All other authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, the enzymatic hydrolysates of skipjack tuna, Katsuwonus pelamis, were purified by ultrafiltration and further identified through micro-ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (micro-UPLC-QTOF-MS). The potential umami peptides were identified using both conventional collision-induced dissociation (CID) and novel electron-activated dissociation (EAD) fragmentation techniques. Nine novel umami peptides with iUmami-SCM > 588 were screened. Sensory evaluation and electronic tongue analysis were performed to confirm the taste characteristics of the umami peptides, indicating that these umami peptides all exhibited varying degrees of umami taste. Molecular docking and molecular dynamics simulation were utilized to investigate the interaction with T1R1/T1R3 taste receptors. The docking results revealed that Asp234, Ser23, Glu231, and Ile237 appeared most frequently in all docking sites and formed stable complexes through hydrogen bonding and electrostatic interactions. Furthermore, molecular dynamics simulation allowed for a more comprehensive analysis of their interactions within a dynamic environment, providing a deeper understanding of the umami perception mechanism involving umami peptides and receptors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study explores the potential of natural bioactive peptides from animal venoms as targeted anti-cancer agents with reduced toxicity. Initially, we screened a broad collection of animal venoms for their antiproliferative activity against cancer cell lines. From this collection, we selected venoms from Naja anchietae and Naja senegalensis due to their promising activity. Utilizing reverse- phase high-performance liquid chromatography (RP HPLC), mass spectrometry (MALDI-TOF MS and MALDI-TOF TOF MSMS), and Edman degradation sequencing, we isolated and characterized three peptides named CTNanc1, CTNanc2, and CTNanc3 from Naja anchietae , and three others named CTNsen1, CTNsen2, and CTNsen3 from Naja senegalensis , each with a molecular weight of around 7 kDa. These purified peptides demonstrated inhibition of U87 glioblastoma cell proliferation, but not of U251 and T98G cells, in cell viability assays. To assess the impact of these treatments on cell viability, apoptosis, and necrosis, flow cytometry assays were conducted on U87 cells at 72 h. The results showed a decrease in cell viability and an increase in dead cells, suggesting that the treatments not only promote apoptosis, but may also lead to increased necrosis or late-stage apoptosis as the exposure time increases. These findings suggest that these peptides could be developed as leads for cancer therapy.

Abalone is a rich source of nutrition, the viscera of which are discarded as by-product during processing. This study explored the biological activities of peptides derived from abalone viscera (AV). Trypsin-hydrolysate of AV (TAV) was purified into three fractions using a Sephadex G-10 column. Nine bioactive peptides (VAR, NYER, LGPY, VTPGLQY, QFPVGR, LGEW, QLQFPVGR, LDW, and NLGEW) derived from TAV-F2 were sequenced. LGPY, VTPGLQY, LGEW, LDW, and NLGEW exhibited antioxidant properties, with IC 50 values of 0.213, 0.297, 0.289, 0.363, and 0.303 mg/mL, respectively. In vitro analysis determined that the peptides VAR, NYER, VTPGLQY, QFPVGR, LGEW, QLQFPVGR, and NLGEW inhibited ACE, with IC 50 values of 0.104, 0.107, 0.023, 0.023, 0.165, 0.004, and 0.146 mg/mL, respectively. The binding interactions of ACE-bioactive peptide complexes were investigated using docking analysis with the ZDCOK server. VTPGLQT interacted with HIS513 and TYR523, and QLQFPVGR interacted with HIS353, ALA354, GLU384, HIS513, and TYR523, contributing to the inhibition of ACE activity. They also interacted with amino acids that contribute to stability by binding to zinc ions. QFPVGR may form complexes with ACE surface sites, suggesting indirect inhibition. These results indicate that AV is a potential source of bioactive peptides with dual antioxidant and anti-hypertensive dual effects.

The increasing awareness of environmental issues and the transition to green analytical chemistry (GAC) have gained popularity among academia and industry in recent years. One of the principles of GAC is the reduction and replacement of toxic solvents with more sustainable and environmentally friendly ones. This review gives an overview of the advances in applying green solvents as an alternative to the traditional organic solvents for peptide and protein purification and analysis by liquid chromatography (LC) and capillary electrophoresis (CE) methods. The feasibility of using greener LC and CE methods is demonstrated through several application examples; however, there is still plenty of room for new developments to fully realize their potential and to address existing challenges. Thanks to the tunable properties of designer solvents, such as ionic liquids and deep eutectic solvents, and almost infinite possible mixtures of components for their production, it is possible that some new designer solvents could potentially surpass the traditional harmful solvents in the future. Therefore, future research should focus mainly on developing new solvent combinations and enhancing analytical instruments to be able to effectively work with green solvents., (© 2024 Wiley‐VCH GmbH.)

Background: Garlic is a promising source of antimicrobial peptide separation, and chemical modification is an effective method for activity improvement. The present study aimed to improve the antifungal activity of a peptide extracted from garlic. Chemical modifications were conducted, and the structure-activity relationship and antifungal mechanism were investigated., Results: The results indicated that the cationic charge induced by Lys residue at the N-terminal was important for the antimicrobial activity, and the modified sequence exhibited significant antifungal activity with low mammalian toxicity and a low tendency of drug resistance (p < 0.05). The structure-activity relationship analysis revealed that the modified active peptide had a predominant α-helical structure and an inner cyclic correlation. Transcriptomic analysis showed that peptide KMLKKLFR (Lys-Met-Leu-Lys-Lyse-Leu-Phe-Arg) affected the rRNA processing and carbon metabolism process of Candida albicans. In addition, the membrane potential study indicated a non-membrane destruction mechanism, and molecular docking analysis and a DNA interaction assay suggested promising inner targets., Conclusion: The results of the present study indicate that chemical modification by amino acid substitution was effective for antimicrobial activity improvement. The present study would benefit future antimicrobial peptide development and suggests that garlic is a great source of antibacterial peptides and peptide template separations for coping with antibiotic resistance. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

This study investigated antioxidant and anti-inflammatory peptides from Edible Bird's Nest (EBN). The prepared EBN peptides were sequentially separated, purified, and successively identified by ultrafiltration, gel filtration and mass spectrometry techniques. Four potential antioxidant and anti-inflammatory peptides were identified as Peptide 1 (LFWSPSVYLK), Peptide 2 (GWPHLEDNYLDW), Peptide 3 (NPPADLHK) and Peptide 4 (GDLAYLDQGHR). Molecular docking analysis revealed that Peptide 1 and Peptide 2 can competitively interrupt the formation of Keap1-Nrf2 due to the presence of hydrophobic and antioxidant amino acids in their peptide sequences. Peptide 3 and Peptide 4 have a strong effect on interacting with the binding site of IKK-β due to the interaction of anti-inflammatory amino acids and C-terminal arginine/lysine. The four peptides were synthesised and validated for their antioxidant and anti-inflammatory activities. The results suggest that the four peptides may serve as promising bioactive peptides for preventing oxidative stress and inflammation-related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recent research has demonstrated the increasing interest in using insects for the extraction of bioactive compounds, particularly peptides. These compounds offer a spectrum of beneficial physiological effects. The aim of this study was to standardize a methodology for obtaining bioactive peptides from Tenebrio molitor and evaluate its physicochemical characterization, antioxidant, and antimicrobial potential. Six assays were carried out to hydrolyse larvae protein, with variations in Alcalase concentration (0.04 to 0.08%) and reaction time (3 to 8 h). The results indicated that the process applied to defatted mealworm flour was effective in reducing lipids by 82.5%. Consequently, it was an observed increase of 38.4% in protein content. Additionally, an increase in glycogen content was found in defatted mealworm flour (177 µmol glucose g-1 sample) and peptides (152.81 µmol glucose g-1 sample). The degree of hydrolysis was higher in assays with longer hydrolysis durations (8.14 - 8.38%). The antioxidant capacity was 12 to 14% lower in assays with an incubation time of 8h. In this sense, the methodology proposed in the present study proved to be efficient in obtaining bioactive peptides from T. molitor.

Recently, jellyfish venom has gained attention as a promising reservoir of pharmacologically active compounds, with potential applications in new drug development. In this investigation, novel peptides, isolated from the hydrolysates of Nemopilema nomurai jellyfish venom (NnV), demonstrate potent inhibitory activities against angiotensin-converting enzyme (ACE). Proteolytic enzymes-specifically, papain and protamex-were utilized for the hydrolysis under optimized enzymatic conditions, determined by assessing the degree of hydrolysis through the ninhydrin test. Comparative analyses revealed that papain treatment exhibited a notably higher degree of NnV hydrolysis compared to protamex treatment. ACE inhibitory activity was quantified using ACE kit-WST, indicating a substantial inhibitory effect of 76.31% for the papain-digested NnV crude hydrolysate, which was validated by captopril as a positive control. The separation of the NnV-hydrolysate using DEAE sepharose weak-anion-exchange chromatography revealed nine peaks under a 0-1 M NaCl stepwise gradient, with peak no. 3 displaying the highest ACE inhibition of 96%. The further purification of peak no. 3 through ODS-C18 column reverse-phase high-performance liquid chromatography resulted in five sub-peaks (3.1, 3.2, 3.3, 3.4, and 3.5), among which 3.2 exhibited the most significant inhibitory activity of 95.74%. The subsequent analysis of the active peak (3.2) using MALDI-TOF/MS identified two peptides with distinct molecular weights of 896.48 and 1227.651. The peptide sequence determined by MS/MS analysis revealed them as IVGRPLANG and IGDEPRHQYL. The docking studies of the two ACE-inhibitory peptides for ACE molecule demonstrated a binding affinity of -51.4 ± 2.5 and -62.3 ± 3.3 using the HADDOCK scoring function.

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC 50 value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs.

The retention of three peptides was studied under analytical and overloaded conditions at different concentrations of trifluoroacetic acid (TFA) and water added to the co-solvent methanol (MeOH). Four columns with different stationary phase properties, i.e., silica, diol, 2-ethylpyridine and cyanopropyl (CN) columns, were evaluated in this investigation. The overall aim was to get a deeper understanding on how column chemistry as well as water and TFA in the co-solvent affect the analytical and overloaded elution profiles using multivariate design of experiments and adsorption measurements of co-solvent components. Multivariate experimental design modeling indicated that water had on average around five times higher effect on the retention than the addition of TFA. The results also showed that the retention increases with the addition of TFA and water to the co-solvent on all columns except the CN column, on which the retention decreased. When examining the effect of adding water to the co-solvent, evidence of a hydrophilic interaction liquid chromatography (HILIC)-like retention mechanism was found on the three other columns with more polar stationary phases. However, on the CN column water acted as an additive, decreasing the retention due to competition with the peptide for available adsorption surface. Adsorption isotherm measurements of the polar solvent MeOH showed that MeOH adsorbs much weaker on the CN column than on the other columns. Addition of TFA and water to the co-solvent substantially sharpened the elution profiles under both overloaded and analytical conditions. Adding a small amount of TFA (from 0 % to 0.05 %) to the co-solvent substantially improved the peak shape of the elution profiles, while further addition (from 0.05 % to 0.15 %) had only a minor effect on the elution profile shape. The reduced retention on the CN column could not be explained by TFA adsorption, which was very weak on all studied columns (retention factor, 0.05-0.15). One could therefore speculate that the ion-pairing complex formed between the peptide and TFA in the mobile phase, reduce the retention due to its reduced polarity. On the other columns displaying HILIC-like properties, the TFA probably just decreased the pH of the mobile phase, thereby promoting the partitioning of the peptide into the water-rich layer. Finally, peak deformation due to diluent-eluent mismatch was observed under overloaded conditions. This was most severe in the cases where MeOH adsorption to the stationary phase was strong and the peptides were only mildly retained. Adding 1,4-dioxan to the diluent resolved this issue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

This study employed a diverse approach to extract antioxidant peptides from red seaweed Palmaria palmata , recognized for its comparatively high protein content. Initially, an aqueous extraction of the entire seaweed was performed, followed by enzymatic hydrolysis of the solid residues prepared from the first step. The effects of three different pH levels (3, 6, and 9) during the aqueous extraction were also examined. Results indicated that the solid fraction from the sequential extraction process contained significantly higher levels of proteins and amino acids than other fractions ( p < 0.05). Furthermore, the solid fractions (IC 50 ranging from 2.29 to 8.15 mg.mL -1 ) demonstrated significantly greater free radical scavengers than the liquid fractions (IC 50 ranging from 9.03 to 10.41 mg.mL -1 or not obtained at the highest concentration tested) at both stages of extraction ( p < 0.05). Among the solid fractions, those produced fractions under alkaline conditions were less effective in radical scavenging than the produced fractions under acidic or neutral conditions. The fractions with most effective metal ion chelating activity were the solid fractions from the enzymatic stage, particularly at pH 3 (IC 50 = 0.63 ± 0.04 mg.mL -1 ) and pH 6 (IC 50 = 0.89 ± 0.07 mg.mL -1 ), which were significantly more effective than those from the initial extraction stage ( p < 0.05). Despite no significant difference in the total phenolic content between these solid fractions and their corresponding liquid fractions (3.79 ± 0.05 vs. 3.48 ± 0.02 mg.mL -1 at pH 3 and 2.43 ± 0.22 vs. 2.51 ± 0.00 mg.mL -1 at pH 6) ( p > 0.05), the observed antioxidant properties may be attributed to bioactive amino acids such as histidine, glutamic acid, aspartic acid, tyrosine, and methionine, either as free amino acids or within proteins and peptides.

Jack bean (JB), Canavalia ensiformis (L.) DC, is a commonly cultivated legume in Indonesia. It is rich in protein, which can be hydrolyzed, making it potentially a good source of bioactive peptides. Intestinal inflammation is associated with several diseases, and the production of interleukin-8 (IL-8) in intestinal epithelial cells induced by tumor necrosis factor (TNF)-α has an important role in inflammatory reaction. The present study investigated the anti-inflammatory effects of peptides generated from enzymatic hydrolysis of JB protein on human intestinal Caco-2BBe cells. Additionally, in silico approaches were used to identify potential bioactive peptides. JB protein hydrolysate (JBPH) prepared using pepsin and pancreatin reduced the IL-8 expression at protein and mRNA levels in Caco-2BBe cells stimulated with TNF-α. Immunoblot analysis showed that the JBPH reduced the TNF-α-induced phosphorylation of c-Jun-NH(2)-terminal kinase, nuclear factor kappa B (NF-κB), and p38 proteins. Anti-inflammatory activity was observed in the 30% acetonitrile fraction of JBPH separated on a Sep-Pak C18 column. An ultrafiltration method revealed that relatively small peptides (< 3 kDa) had a potent inhibitory effect on the IL-8 production. Purification of the peptides by reversed-phase and anion-exchange high performance chromatography produced three peptide fractions with anti-inflammatory activities. A combination of mass spectrometry analysis and in silico approaches identified the potential anti-inflammatory peptides. Peptides derived from JB protein reduces the TNF-α-induced inflammatory response in Caco-2BBe cells via NF-κB and mitogen-activated protein kinase signaling pathways. Our results may lead to a novel therapeutic approach to promote intestinal health., (© 2024. The Author(s).)

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 μg/mg to 7.94 ± 1.56 μg/mg. Peptides with a molecular weight of 1-2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to β-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide‑calcium complexes. The F21 contained 42 peptides, with repeated "GE" motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study isolated a novel antioxidant peptide from black soldier fly larvae (BSFL) using enzymatic hydrolysis. Firstly, the BSFL enzymatic hydrolysate was fractionated through ultrafiltration, with the <3 kDa fraction exhibiting the strongest DPPH and ABTS radical scavenging activity. Subsequently, this fraction was further fractionated through gel filtration chromatography and RP-HPLC. Totally, 153 peptides were identified through LC-MS/MS analysis, from which a novel peptide EDEGTYKCVLS (Pep6) was screened according to activity prediction and verification. Pep6 exhibited high radical scavenging capacity and cytoprotective effect on HepG2 cells against H 2 O 2 damage, meanwhile significantly increasing the intracellular antioxidant enzymes activity. Molecular docking analysis indicated that Pep6 competitively bound to Keap1, thereby inhibiting the formation of Keap1-Nrf2 complex, ultimately protecting cells from oxidative stress damage. In this study, a novel antioxidant peptide Pep6 was identified from BSFL, and its antioxidant mechanism was elucidated, providing a theoretical basis for its use as a natural antioxidant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Context: Plant peptides garner attention for their potential antimicrobial properties amid the rising concern over antibiotic-resistant bacteria., Objective: This study investigates the antibacterial potential of crude peptide extracts from 27 Thai plants collected locally., Materials and Methods: Peptide extracts from 34 plant parts, derived from 27 Thai plants, were tested for their antimicrobial efficacy against four highly resistant bacterial strains: Streptococcus aureus MRSA, Pseudomonas aeruginosa , Acinetobacter baumannii , and Escherichia coli . The stability of these peptide extracts was examined at different temperatures, and the synergistic effects of two selected plant peptide extracts were investigated. Additionally, the time-kill kinetics of the individual extracts and their combination were determined against the tested pathogens., Results: Peptides from Allium sativum L. and Allium oschaninii O. Fedtsch (Amaryllidaceae) were particularly potent, inhibiting bacterial growth with MICs ranging from 1.43 to 86.50 µg/mL. The consistent MICs and MBCs of these extracts across various extraction time points highlight their reliability. Stability tests reveal that these peptides maintain their antimicrobial activity at -20 °C for over a month, emphasizing their durability for future exploration and potential applications in addressing antibiotic resistance. Time-kill assays elucidate the time and concentration-dependent nature of these antimicrobial effects, underscoring their potent initial activity and sustained efficacy over time., Discussion and Conclusions: This study highlights the antimicrobial potential of Allium -derived peptides, endorsing them for combating antibiotic resistance and prompting further investigation into their mechanisms.

Background: Peanut peptides can chelate iron but their chelation mechanism remains unclear. The purpose of this study is to separate peanut ferrous-chelating peptides and explore the chelation mechanism of peanut peptides with iron., Results: Peanut peptide component F-122, which had a higher chelation rate, was separated using ultrafiltration, gel filtration chromatography, and ion exchange chromatography, achieving a ferrous chelation rate of 90.7%. Six peptide segments were screened and their amino acid sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectral analysis confirmed that the chelation between peanut peptides and ferrous ions occurred and a new substance was formed. Molecular docking simulation indicated that the amino acids in peanut peptides involved in the chelating reaction were glutamic acid, arginine, glycine, threonine, phenylalanine, and lysine. The binding sites included the main chain oxygen atom, side chain oxygen atom, and carboxyl oxygen atom of amino acid., Conclusion: The isolated peanut peptide had a higher ferrous-chelation rate. The chelating mechanism of peanut peptide with ferrous ion was elucidated. This study provides a theoretical basis for the development of new peptide-ferrous preparations. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Angiotensin I-converting enzyme (ACE) regulates blood pressure through the renin-angiotensin system. Douchi, a traditional fermented soybean condiment, may have antihypertensive effects, but research on ACE inhibitory peptides from Douchi hydrolysates is limited. We hypothesized that enzymatic treatment could enhance ACE inhibitory peptide diversity and efficacy. We tested ten single enzymes and four combinations, finding pepsin-trypsin-chymotrypsin most effective. Hydrolysates were purified using Sephadex G-15 and reversed-phase HPLC, and peptides were identified via LC-MS/MS. Five peptides (LF, VVF, VGAW, GLFG, NGK) were identified, with VGAW as the most potent ACE inhibitor (IC 50 46.6 ± 5.2 μM) showing excellent thermal and pH stability. Lineweaver-Burk plots confirmed competitive inhibition, and molecular docking revealed eight hydrogen bonds between VGAW and ACE. In hypertensive rats, VGAW significantly reduced blood pressure at 12.5, 25, and 50 mg/kg. These findings highlight Douchi as a source of ACE inhibitory peptides and suggest VGAW as a promising functional food ingredient., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The effect of the released amount and bitterness threshold of bitter peptides on the sensory properties of different wheat gluten hydrolysates (WGHs) after hydrolysis was investigated. The results showed that the endo-activity of the enzyme promoted the release of bitter peptides, leading to enhanced bitterness intensity in WGHs. With the increase in degree of hydrolysis (DH), the bitter threshold of bitter peptides became the main reason affecting bitterness of the WGHs. Proteax exerted the strong exo-activity at the DH of 20%, which could reduce bitterness of Pro-16 hydrolysates. The reason for debittering was the reduction in the content with molecular weights (MWs) of 500-1000 Da and the decrease of surface hydrophobicity (SH) in the Pro-20 M hydrolysates, which led to the increase of the bitterness threshold of bitter peptide. Meanwhile, HPLC-MS/MS analysis demonstrated the reduced proportion of C-terminal hydrophobic amino acids (HAAs) in Pro-20 M extracts verifying the cause of debittering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ulva prolifera , a type of green algae that can be consumed, was utilized in the production of an angiotensin-I converting enzyme (ACE) inhibitory peptide. The protein from the algae was isolated and subsequently hydrolyzed using a neutral protease. The resulting hydrolysate underwent several processes including Sephadex-G100 filtration chromatography, ultrafiltration, HPLC-Q-TOF-MS analysis, ADMET screening, UV spectrum detection test, molecular docking, and molecular dynamic simulation. Then, the ACE inhibitory peptide named KAF (IC 50 , 0.63 ± 0.26 µM) was identified. The effectiveness of this peptide in inhibiting ACE can be primarily attributed to two conventional hydrogen bonds. Additionally, it could activate endothelial nitric oxide synthase (eNOS) activity to promote the generation of nitric oxide (NO). Additionally, KAF primarily increased the intracellular calcium (Ca 2+ ) level by acting on L-type Ca 2+ channel (LTCC) and the ryanodine receptor (RyR) in the endoplasmic reticulum, and completed the activation of eNOS under the mediation of protein kinase B (Akt) signaling pathway. Our study has confirmed that KAF has the potential to be processed into pharmaceutical candidate functions on vasoconstriction.

With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG) 3 . Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The precise determination of polypeptide antibiotics (PPTs) in foods has been always challenging because of the interference of various endogenous peptides in complex matrix. Herin, a novel large-pore covalent organic framework (TABPT-SPDA-COF) with accessible pore size of 7.9 nm was synthesized as a solid phase extraction (SPE) absorbent for efficiently enriching four PPTs existed in foods originating from animals. The parameters of SPE process were systematically optimized. Subsequently, four PPTs were determined by UHPLC-MS/MS. Under the optimal conditions, TABPT-SPDA-COF shows outstanding enrichment capacity for PPTs in contrast to commercial absorbents ascribed to size selectivity and multiple interaction effects. The method exhibits excellent linear range (0.005-100 ng mL -1 ), satisfactory limits of detection (0.1 pg mL -1 ) as well as relative recoveries (86.2-116 %). This work offers a practicable platform to monitor trace PPTs from complex animal-derived foodstuffs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The inhibition of dipeptidyl peptidase-IV (DPP-IV) is a promising approach for regulating the blood glucose levels in patients with type 2 diabetes (T2D). Oysters, rich in functional peptides, contain peptides capable of inhibiting DPP-IV activity. This study aims to identify the hypoglycemic peptides from oysters and investigate their potential anti-T2D targets and mechanisms. This research utilized virtual screening for the peptide selection, followed by in vitro DPP-IV activity assays to validate the chosen peptide. Network pharmacology was employed to identify the potential targets, GO terms, and KEGG pathways. Molecular docking and molecular dynamics simulations were used to provide virtual confirmation. The virtual screening identified LRGFGNPPT as the most promising peptide among the screened oyster peptides. The in vitro studies confirmed its inhibitory effect on DPP-IV activity. Network pharmacology revealed that LRGFGNPPT exerts an anti-T2D effect through multiple targets and signaling pathways. The key hub targets are AKT1, ACE, and REN. Additionally, the molecular docking results showed that LRGFGNPPT exhibited a strong binding affinity with targets like AKT1, ACE, and REN, which was further confirmed by the molecular dynamics simulations showcasing a stable peptide-target interaction. This study highlights the potential of LRGFGNPPT as a natural anti-T2D peptide, providing valuable insights for potential future pharmaceutical or dietary interventions in T2D management.

Pearl and nacre powders have been valuable traditional Chinese medicines with whitening properties for thousands of years. We utilized a high-temperature and high-pressure method along with compound enzyme digestion to prepare the enzymatic hydrolysates of nacre powder of Pinctada martensii (NP-PMH). The peptides were identified using LC-MS/MS and screened through molecular docking and molecular dynamics simulations. The interactions between peptides and tyrosinase were elucidated through enzyme kinetics, circular dichroism spectropolarimetry, and isothermal titration calorimetry. Additionally, their inhibitory effects on B16F10 cells were explored. The results showed that a tyrosinase-inhibitory peptide (Ala-His-Tyr-Tyr-Asp, AHYYD) was identified, which inhibited tyrosinase with an IC 50 value of 2.012 ± 0.088 mM. The results of the in vitro interactions showed that AHYYD exhibited a mixed-type inhibition of tyrosinase and also led to a more compact enzyme structure. The binding reactions of AHYYD with tyrosinase were spontaneous, leading to the formation of a new set of binding sites on the tyrosinase. The B16F10 cell-whitening assay revealed that AHYYD could reduce the melanin content of the cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that AHYYD could be widely used as a tyrosinase inhibitor in whitening foods and pharmaceuticals.

Data-independent acquisition (DIA) techniques such as sequential window acquisition of all theoretical mass spectra (SWATH) acquisition have emerged as the preferred strategies for proteomic analyses. Our study optimized the SWATH-DIA method using a narrow isolation window placement approach, improving its proteomic performance. We optimized the acquisition parameter combinations of narrow isolation windows with different widths (1.9 and 2.9 Da) on a ZenoTOF 7600 (Sciex); the acquired data were analyzed using DIA-NN (version 1.8.1). Narrow SWATH (nSWATH) identified 5916 and 7719 protein groups on the digested peptides, corresponding to 400 ng of protein from mouse liver and HEK293T cells, respectively, improving identification by 7.52 and 4.99%, respectively, compared to conventional SWATH. The median coefficient of variation of the quantified values was less than 6%. We further analyzed 200 ng of benchmark samples comprising peptides from known ratios of Escherichia coli , yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to those of conventional SWATH, identifying an average of 95,456 precursors and 9342 protein groups across three benchmark samples, representing 12.6 and 9.63% improved identification compared to conventional SWATH. The nSWATH method improved identification at various loading amounts of benchmark samples, identifying 40.7% more protein groups at 25 ng. These results demonstrate the improved performance of nSWATH, contributing to the acquisition of deeper proteomic data from complex biological samples.

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 μg of cell lysate as the starting material. Three thousand seven hundred ninety-six ( N = 2, RSD = 4.95%) protein groups and 20,577 ( N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.

This study investigated the capability of electromembrane extraction (EME) as a general technique for peptides, by extracting complex pools of peptides comprising in total of 5953 different substances, varying in size from seven to 16 amino acids. Electromembrane extraction was conducted from a sample adjusted to pH 3.0 and utilized a liquid membrane consisting of 2-nitrophenyl octyl ether and carvacrol (1:1 w/w), containing 2% (w/w) di(2-ethylhexyl) phosphate. The acceptor phase was 50 mM phosphoric acid (pH 1.8), the extraction time was 45 min, and 10 V was used. High extraction efficiency, defined as a higher peptide signal in the acceptor than the sample after extraction, was achieved for 3706 different peptides. Extraction efficiencies were predominantly influenced by the hydrophobicity of the peptides and their net charge in the sample. Hydrophobic peptides were extracted with a net charge of +1, while hydrophilic peptides were extracted when the net charge was +2 or higher. A computational model based on machine learning was developed to predict the extractability of peptides based on peptide descriptors, including the grand average of hydropathy index and net charge at pH 3.0 (sample pH). This research shows that EME has general applicability for peptides and represents the first steps toward in silico prediction of extraction efficiency., (© 2024 The Author(s). Journal of Separation Science published by Wiley‐VCH GmbH.)

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with K d estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hypsizygus marmoreus has abundant proteins and is a potential source for the development of bioactive peptides. However, currently, the research on the bioactive components of H. marmoreus mainly focuses on polysaccharides, and there is no relevant research on the preparation of bioactive peptides. In this article, an ultrasound-assisted extraction method was used to extract proteins from H. marmoreus , and then, four peptides with different molecular weight ranges were prepared through protease hydrolysis and molecular classification. The antioxidant and antibacterial activities were also studied. Under the optimal conditions, the extraction rate of H. marmoreus proteins was 53.6%. Trypsin exhibited the highest hydrolysis rate of H. marmoreus proteins. The optimal parameters for enzymatic hydrolysis were a substrate concentration of 3.7%, enzyme addition of 5700 U/g, pH value of 7, extraction temperature of 55 °C, and time of 3.3 h. Under these conditions, the peptide yield was 59.7%. The four types of H. marmoreus peptides were prepared by molecular weight grading. Among them, peptides with low molecular weight (<1 kDa) had stronger antioxidant and antibacterial activities. This study provides a theoretical basis for the efficient preparation of H. marmoreus peptides and the development of antioxidant and antibacterial peptide products.

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans ( Phaseolus vulgaris ). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O -glycan within these peptides.

Hairy tofu is a famous Chinese snack that is made from soybeans and rich in various nutrients. In order to further explore the antioxidant peptides of hairy tofu hydrolysates, seven proteases were used to hydrolyze hairy tofu. The results of in vitro radical scavenging activity showed that hairy tofu hydrolysates obtained by pancreatin exhibited the highest antioxidant activity. After Sephadex G-25 gel filtration and reversed-phase high-performance liquid chromatography (RP-HPLC), 97 peptides were identified in the most antioxidant fraction using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Among them, nine peptides were synthesized and their antioxidant activities were assessed using a H 2 O 2 -induced oxidative 293T cell model. Finally, four peptides (QCESHK, LAWNEGR, NLQGENEWDQK, and FTEMWR) at concentrations of < 50 μg/ml significantly decreased the malondialdehyde content compared with the model group, displaying in vivo antioxidant activity and low cytotoxicity. Overall, this research provided the choice of using hairy tofu peptides as antioxidant products in the pharmaceutical and food industries., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

This study generated bioactive hydrolysates using the enzyme Alcalase and autolysis from mesopelagic fish, including Maurolicus muelleri and Benthosema glaciale . Generated hydrolysates were investigated for their bioactivities using in vitro bioassays, and bioactive peptides were identified using mass spectrometry in active hydrolysates with cyclooxygenase, dipeptidyl peptidase IV and antioxidant activities. In silico analysis was employed to rank identified peptide sequences in terms of overall bioactivity using programmes including Peptide Ranker, PrepAIP, Umami-MRNN and AntiDMPpred. Seven peptides predicted to have anti-inflammatory, anti-type 2 diabetes or Umami potential using in silico strategies were chemically synthesised, and their anti-inflammatory activities were confirmed using in vitro bioassays with COX-1 and COX-2 enzymes. The peptide QCPLHRPWAL inhibited COX-1 and COX-2 by 82.90% (+/-0.54) and 53.84%, respectively, and had a selectivity index greater than 10. This peptide warrants further research as a novel anti-inflammatory/pain relief peptide. Other peptides with DPP-IV inhibitory and Umami flavours were identified. These offer potential for use as functional foods or topical agents to prevent pain and inflammation.

Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Å fully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Å pore size materials─was more pronounced for smaller peptides. These findings contradict the central dogmas that underlie the development of all peptide RP-HPLC applications: (i) a larger surface area leads to higher retention and (ii) increasing the pore size should benefit the retention of larger analytes. Based on our intriguing findings, we compared reversed-phase high-performance liquid chromatography peptide retention for a total of 20 columns with pore sizes between 60 and 300 Å using FA- and TFA-based eluents. Our results unequivocally attest that the larger size of ion pairs in FA- vs TFA-based eluents leads to the observed impact on selectivity and peptide retention. For FA, peptide retention peaks at 200 Å pore size, compared to between 120 and 200 Å for TFA. However, the decrease in retention for narrow-pore particles is more profound in FA. Our findings suggest that common assumptions about analyte size and accessible surface area should be revisited for ion-pair RP separation of small peptides, typical for proteomic applications that are predominantly applying FA eluents. Hybrid silica-based materials with pore sizes of 130-200 Å should be specifically targeted for bottom-up proteomic applications to obtain both superior peak shape and peptide retentivity. This challenging task of attaining the best RPLC column for proteomics calls for closer collaboration between LC column manufacturers and proteomic LC specialists.

Bioprospecting the secondary metabolism of underexplored Actinomycetota taxa is a prolific route to uncover novel chemistry. In this work, we report the isolation, structure elucidation, and bioactivity screening of cellulamides A and B ( 1 and 2 ), two novel linear peptides obtained from the culture of the macroalga-associated Cellulosimicrobium funkei CT-R177. The host of this microorganism, the Chlorophyta Codium tomentosum , was collected in the northern Portuguese coast and, in the scope of a bioprospecting study focused on its associated actinobacterial community, strain CT-R177 was isolated, taxonomically identified, and screened for the production of antimicrobial and anticancer compounds. Dereplication of a crude extract of this strain using LC-HRMS(/MS) analysis unveiled a putative novel natural product, cellulamide A ( 1 ), that was isolated following mass spectrometry-guided fractionation. An additional analog, cellulamide B ( 2 ) was obtained during the chromatographic process and chemically characterized. The chemical structures of the novel linear peptides, including their absolute configurations, were elucidated using a combination of HRMS, 1D/2D NMR spectroscopy, and Marfey's analysis. Cellulamide A ( 1 ) was subjected to a set of bioactivity screenings, but no significant biological activity was observed. The cellulamides represent the first family of natural products reported from the Actinomycetota genus Cellulosimicrobium , showcasing not only the potential of less-explored taxa but also of host-associated marine strains for novel chemistry discovery.

Parmotrema perlatum, a lichen belonging to the family Parmeliaceae, is well known for its culinary benefits and aroma used as a condiment in Indian homes is also known as the "black stone flower" or "kalpasi" in India. This research intends to analyze the antioxidant power of the crude extracts using four pH-based buffers solubilized proteins/peptides and RP-HPLC fractions of P. perlatum obtained by purification. The proteins that were extracted from the four different buffers were examined using LC-MS/MS-based peptide mass fingerprinting. When compared to the other buffers, the 0.1 M of Tris-HCl buffer pH 8.0 solubilized proteins/peptides had the strongest antioxidant capacity. The sequential purification of the peptide was carried out by using a 3-kDa cut-off membrane filter and semipreparative RP-HPLC. Additionally, the purified fractions of the peptide's antioxidant activity were assessed, and effects were compared with those of the crude and 3 kDa cut--off membrane filtrates. The peptide fractions were sequenced by LC-MS/MS, which reveals that fraction 2 from RP-HPLC with the sequence LSWFMVVAP has shown the highest antioxidant potential in comparison with other fractions which can serve as the potential natural antioxidant drug. Further, fraction 2 also showed antibacterial activity against the selected microorganisms., (© 2024 International Union of Biochemistry and Molecular Biology, Inc.)

A combination of LC-MS/MS and feature-based molecular networking analyses led to the isolation of a new adenopeptin analog, higapeptin (1), and four known peptides, adenopeptin (2), adenopeptins B and C (3 and 4), and acremopeptin (5), from the rice culture of the fungus Acremonium persicinum (18F04103) isolated from a mud flat of the Ariake Sea in Kyushu, Japan. The structure of 1 was determined by NMR and MS/MS fragmentation analyses. The absolute configuration of the constituent amino acids was determined by Marfey's analysis after acid hydrolysis. The C-terminal residue was synthesized, and its absolute configuration was established by Marfey's analysis. Compounds 1 and 2 were found to inhibit mitochondrial energy metabolism, similar to efrapeptin D (6), a known mitochondrial ATPase inhibitor., (© 2024. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders. The specific isolation and purification of intact, high-purity, and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks. In this study, a mitochondrial penetrating peptide (MPP) with the sequence FrFKFrFK(Ac) was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material. The multiple aromatic phenylalanine (F) residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane, whereas the basic residues (D-arginine and lysine) render the MPP surface positively charged, thereby promoting the binding of negatively charged mitochondria. After the derivatization of the N terminal of MPP with an oligoglycine spacer, the peptide ligands were conjugated to matrix beads (MB) with surface aldehyde functional groups. Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads. The generated Schiff bases were reduced, affording stable covalent bonds. The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography (HPLC), zeta potential assay, and scanning electron microscopy (SEM). The immobilization efficiency of the peptide ligands was 1.47 μmol/g, and the surface potential of MB@MPP was 11 mV. MB@MPP was used for the direct isolation of mitochondria after cell homogenization. As observed by SEM, mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface. Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes, the specific binding and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions. Western blot assays were employed to characterize the protein markers of the mitochondria (citrate synthase (CS) and voltage-dependent anion channel protein (VDAC)) and cytoplasmic protein (vinculin), and examine the integrity and purity of the captured mitochondria. The results showed that the lysates released from MB@MPP had high CS and VDAC contents. By contrast, vinculin, which is highly abundant in whole-cell lysates, was barely detected in the lysates from MB@MPP. These results suggest that MB@MPP isolates mitochondria with high affinity, specificity, and antifouling ability by using the targeting peptide as the capture handle. A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity. Given the superior separation performance of MB@MPP, the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to detect tryptophan (Trp) and riboflavin in the mitochondria. Quantification was performed in multiple-reaction monitoring (MRM) mode. Owing to the high purity of the mitochondria, the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg, respectively. The metabolic response of mitochondria to external stimuli was further examined using acadesine, an adenosine 5'-monophosphate (AMP)-activated protein kinase activator with a wide range of metabolic effects, to treat cells. After cell homogenization, MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment, followed by LC-MS/MS analysis. The quantification results demonstrated that acadesine induced a 14% upregulation of Trp content in the mitochondria. By contrast, the riboflavin content decreased to 0.48 nmol/mg, which is 72% of that in untreated mitochondria. The changes in Trp and riboflavin contents could influence their metabolic pathways and, thus, the levels of their metabolites, such as nicotinamide adenine dinucleotide, flavin mononucleotide, and flavin adenine dinucleotide, which are essential coenzymes in mitochondria. Peptide-functionalized affinity microbeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria, and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.

Recently, there has been a growing interest in collagen peptides derived from marine sources for their notable ability to protect skin cells against apoptosis induced by oxidants. Therefore, the current study aimed to investigate the fundamental properties of collagen peptides, including their physicochemical, thermal, structural, stem-cell-regenerative, and skin-cell-protective effects, in comparison to commercial collagen peptides. The acid-soluble (ASC) and pepsin-soluble (PSC) collagens exhibited three distinct bands on SDS-PAGE, namely α (α 1 and α 2 ), β, and γ chains, confirming a type I pattern. The thermal profiles obtained from TG and DSC analyses confirmed the denaturation of PSC and ASC at temperatures ranging from 51.94 to 56.4 °C and from 52.07 to 56.53 °C, respectively. The purified collagen peptides were analyzed using SDS-PAGE and MALDI-TOF mass spectrometry, revealing a mass range of 900-15,000 Da. Furthermore, the de novo peptide sequence analysis confirmed the presence of the Gly-X-Y repeating sequence in collagen peptides. Collagen peptide treatments significantly enhanced HFF-1 cell proliferation and migration compared to the control group. ELISA results confirmed the potential interactions between collagen peptides and HFF-1 cells through α 2 β 1 , α 10 β 1 , and α 11 β 1 integrin receptors. Notably, collagen peptide treatment effectively restored the proliferation of HFF-1 cells damaged by H 2 O 2 . Consequently, the advantageous characteristics of squid skin collagen peptides highlight their promising role in regenerative medicine.

The skin of fish is a physicochemical barrier that is characterized by being formed by cells that secrete molecules responsible for the first defense against pathogenic organisms. In this study, the biological activity of peptides from mucus of Seriola lalandi and Seriolella violacea were identified and characterized. To this purpose, peptide extraction was carried out from epidermal mucus samples of juveniles of both species, using chromatographic strategies for purification. Then, the peptide extracts were characterized to obtain the amino acid sequence by mass spectrometry. Using bioinformatics tools for predicting antimicrobial and antioxidant activity, 12 peptides were selected that were chemically produced by simultaneous synthesis using the Fmoc-Tbu strategy. The results revealed that the synthetic peptides presented a random coil or extended secondary structure. The analysis of antimicrobial activity allowed it to be discriminated that four peptides, named by their synthesis code 5065, 5069, 5070, and 5076, had the ability to inhibit the growth of Vibrio anguillarum and affected the copepodite stage of C. rogercresseyi . On the other hand, peptides 5066, 5067, 5070, and 5077 had the highest antioxidant capacity. Finally, peptides 5067, 5069, 5070, and 5076 were the most effective for inducing respiratory burst in fish leukocytes. The analysis of association between composition and biological function revealed that the antimicrobial activity depended on the presence of basic and aromatic amino acids, while the presence of cysteine residues increased the antioxidant activity of the peptides. Additionally, it was observed that those peptides that presented the highest antimicrobial capacity were those that also stimulated respiratory burst in leukocytes. This is the first work that demonstrates the presence of functional peptides in the epidermal mucus of Chilean marine fish, which provide different biological properties when the fish face opportunistic pathogens.

Since the outbreak of coronavirus disease 2019, the global demand for vaccines has increased rapidly to prevent infection and protect high-risk populations. However, identifying viral mutations poses an additional challenge for chromatographic purification of vaccines and subunit vaccines. In this study, a new affinity peptide model, X 1 VX 2 GLNX 3 WX 4 RYSK, was established, and a library of 612 peptides was generated for ligand screening. Based on a multistep strategy of ligand screening, 18 candidate peptides were obtained. The top ranking peptide, LP14 (YVYGLNIWLRYSK), and two other representative peptides, LP02 and LP06, with lower rankings were compared via molecular dynamics simulation. The results revealed that peptide binding to the receptor binding domain (RBD) was driven by hydrophobic interactions and the key residues involved in the binding were identified. Surface plasmon resonance analysis further confirmed that LP14 had the highest affinity for the wild RBD (K d =0.520 μmol/L), and viral mutation had little influence on the affinity of LP14, demonstrating its great potential as a broad-spectrum ligand for RBD purification. Finally, chromatographic performance of LP14-coupled gel-packed column verified that both wild and omicron RBDs could be purified and were eluted by 0.1 mol/L Gly-HCl buffer (pH 3.0). This research identified a broad-spectrum peptide for RBD purification based on rational design and demonstrated its potential application in the purification of RBDs from complex feedstock., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)