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Rationale: Heavy-labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)-based bottom-up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times., Methods: We describe an efficient, low-cost optimised method to enable 'in-house' heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC-MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin-catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors., Results: Step-by-step instructions are provided on how to execute this protocol with high-throughput adaptations utilising a 96-well plate and a liquid-handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%., Conclusions: The application of the 'in-house' labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies., (© 2024 The Author(s). Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Development of monoclonal and bispecific antibody-based protein therapeutics requires detailed characterization of native disulfide linkages, which is commonly achieved through peptide mapping under non-reducing conditions followed by liquid chromatography-mass spectrometry (LC-MS) analysis. One major challenge of this method is incomplete protein digestion due to insufficient denaturation of antibodies under non-reducing conditions. For a long time, researchers have explored various strategies with the aim of efficiently digesting antibody drugs when the disulfide bonds remain intact, but few could achieve this by using a simple and generic approach with well controlled disulfide scrambling artifacts. Here, we report a simple method for fast and efficient mapping of native disulfides of monoclonal and bispecific antibody-based protein therapeutics. The method was optimized to achieve optimal digestion efficiency by denaturing proteins with 8 M urea plus 0-1.25 M guanidine-HCl at elevated temperature (50 °C), followed by two-step digestion with trypsin/Lys-C mix using a one-pot reaction. The only parameter that needs to be optimized for different proteins is the concentration of guanidine-HCl present. This simplified sample preparation eliminated buffer exchange and can be completed within three hours. By using this new method, all native disulfide bonds were confirmed for these monoclonal and bispecific antibodies with high confidence. When compared with a commercial kit utilizing low-pH digestion condition, the new method demonstrated higher digestion efficiency and shorter sample preparation time. These results suggest this new one-pot-two-step digestion method is suitable for the characterization of antibody disulfide bonds, particularly for those antibodies with digestion-resistant domains under typical digestion conditions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liqing Gu reports financial support was provided by Incyte Corporation. Tiger X. Hu reports financial support was provided by Incyte Corporation. Liqing Gu reports a relationship with Incyte Corporation that includes: employment and equity or stocks. Tiger X. Hu reports a relationship with Incyte Corporation that includes: employment and equity or stocks., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH 2 (belonging to β-lactoglobulin, position 48-60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved., 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. Nothing declared., (Copyright © 2024. Published by Elsevier Ltd.)

During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology., Competing Interests: The authors declare no conflict of interest.

Serine proteases are among the important groups of enzymes having significant roles in cell biology. Trypsin is a representative member of the serine superfamily of enzymes, produced by acinar cells of pancreas. It is a validated drug target for various ailments including pancreatitis and colorectal cancer. Premature activation of trypsin is involved in the lysis of pancreatic tissues, which causes pancreatitis. It is also reported to be involved in colorectal carcinoma by activating other proteases, such as matrix metalloproteinase (MMPs). The development of novel trypsin inhibitors with good pharmacokinetic properties could play important roles in pharmaceutical sciences. This study reports the crystal structures of bovine pancreatic trypsin with four molecules; cimetidine, famotidine, pimagedine, and guanidine. These compounds possess binding affinity towards the active site (S1) of trypsin. The structures of all four complexes provided insight of the binding of four different ligands, as well as the dynamics of the active site towards the bind with different size ligands. This study might be helpful in designing of new potent inhibitors of trypsin and trypsin like serine proteases., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study explores the synthesis and application of artificial zymogens using protein-polymer hybrids to mimic the controlled enzyme activation observed in natural zymogens. Pro-trypsin (pro-TR) and pro-chymotrypsin (pro-CT) hybrids were engineered by modifying the surfaces of trypsin (TR) and chymotrypsin (CT) with cleavable peptide inhibitors utilizing surface-initiated atom transfer radical polymerization. These hybrids exhibited 70 and 90% reductions in catalytic efficiency for pro-TR and pro-CT, respectively, due to the inhibitory effect of the grafted peptide inhibitors. The activation of pro-TR by CT and pro-CT by TR resulted in 1.5- and 2.5-fold increases in enzymatic activity, respectively. Furthermore, the activated hybrids triggered an enzyme activation cascade, enabling amplification of activity through a dual pro-protease hybrid system. This study highlights the potential of artificial zymogens for therapeutic interventions and biodetection platforms by harnessing enzyme activation cascades for precise control of catalytic activity.

We present receptor hopping and receptor swapping free energy estimation protocols based on the Alchemical Transfer Method (ATM) to model the binding selectivity of a set of ligands to two arbitrary receptors. The receptor hopping protocol, where a ligand is alchemically transferred from one receptor to another in one simulation, directly yields the ligand's binding selectivity free energy (BSFE) for the two receptors, which is the difference between the two individual binding free energies. In the receptor swapping protocol, the first ligand of a pair is transferred from one receptor to another while the second ligand is simultaneously transferred in the opposite direction. The receptor swapping free energy yields the differences in binding selectivity free energies of a set of ligands, which, when combined using a generalized DiffNet algorithm, yield the binding selectivity free energies of the ligands. We test these algorithms on host-guest systems and show that they yield results that agree with experimental data and are consistent with differences in absolute and relative binding free energies obtained by conventional methods. Preliminary applications to the selectivity analysis of molecular fragments binding to the trypsin and thrombin serine protease confirm the potential of the receptor swapping technology in structure-based drug discovery. The novel methodologies presented in this work are a first step toward streamlined and computationally efficient protocols for ligand selectivity optimization between mutants and homologous proteins.

Serum contains several proteins that are associated with disease-related processes. Mass spectrometry (MS)-based proteomics approaches greatly facilitate serum protein biomarker development. However, the serum proteome complexity presents a technical challenge for the accurate, sensitive, and reproducible quantification of proteins by MS. Thus, efficient sample preparation methods are of critical importance for serum proteome analyses. In this study, we evaluated the technical performance of two serum proteome sample preparation methods using sera from patients with high-grade serous ovarian cancer and patients with benign nongynecological conditions with a goal of providing insight into their compatibility with clinical proteomics workflows. One method entailed the use of immobilized trypsin (SMART Digest Trypsin) with RapiGest SF, an acid-labile surfactant designed to enhance the in-solution enzymatic digestion of proteins. The other method incorporated a commercially available sample preparation kit, iST-BCT, which contains standardized reagents. Significantly higher protein sequence coverage, albeit with lower digestion efficiency, was obtained with the immobilized trypsin + RapiGest SF workflow, whereas the iST-BCT workflow was quicker and had marginally better reproducibility. Protein relative abundance analysis revealed that the serum proteomes clustered primarily by the sample processing workflow and secondarily by disease state. We conducted a time course study to determine whether differences in the relative abundance of diagnostic high-grade serous ovarian cancer serum protein biomarker candidates were biased according to the duration of enzymatic digestion. Our results highlight the importance of optimizing enzymatic digestion kinetics according to the peptide targets of interest while considering the sensitivity of the downstream analytical method utilized in clinical proteomics workflows designed to measure biomarkers.

Peptide mapping requires cleavage of proteins in a predictable fashion so that target protein-specific peptides can be reliably identified and quantified. Trypsin, a commonly used protease in this process, can also undergo self-cleavage or autolysis, thereby reducing the effectivity and even cleavage specificity at lysine and arginine residues. Here, we report highly efficient and reproducible peptide mapping of biotherapeutic monoclonal antibodies. We highlight the properties of a homogeneous chemically modified trypsin on thermal stability, a 54% increase in melting temperature with an 84% increase in energy required for unfolding, an indication of more thermally stable trypsin, >90% retained intact mass peak area after exposure to digestion conditions confirming autolysis resistance, 10× more intensity for intact enzyme compared to trypsin of similar source and narrower molecular weight distribution with LC-MS indicative of low degradation compared to 3 other types of trypsin. Finally, we show the utility of this autolysis-resistant trypsin in characterizing biotherapeutic monoclonal antibodies consistently and reliably showing a >30% reduction in missed cleavage for a short-duration protein digestion time of 30 min compared to heterogeneously modified trypsin of a similar source.

In this experiment, the co-constructed O/W emulsions of different soy protein hydrolysates (SPHs) and gum arabic (GA) were investigated. SPHs were prepared by hydrolyzing soy protein isolate (SPI) using different enzymes, and investigated the effects of enzyme types and hydrolysis time on the physicochemical properties of SPHs. Moreover, SPI/GA and SPHs/GA were prepared and used as hydrophilic emulsifiers to construct O/W emulsions. The results showed that the optimal hydrolysis times for bromelain, pepsin and trypsin were 2 h (BSPH2), 3 h (PSPH3) and 3 h (TSPH3), respectively. Compared with SPI/GA emulsions, SPHs/GA emulsions had smaller particle size, more negative charge, higher interfacial adsorbed protein, and more stable emulsion systems. During the digestion process, SPHs/GA emulsions were effective in realizing the release of bioactives. In conclusion, enzymatic hydrolysis can be an effective modification technique, and SPHs/GA can be used as an effective emulsifier for the emulsion system., 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.)

Peptidyl arginine deiminase 4 (PAD4) plays a critical role in many autoimmune diseases including rheumatoid arthritis. Herein, a trypsin assisted highly immunoassay method was established to determine PAD4 activity and screen potent inhibitors from herbal plants extracts and purified natural products. The method was applied to determine endogenous PAD4 activity in both cell and tissue lysates, as well as the inhibitory effects of 20 herbal plants and 50 purified natural products. The Cinnamomi ramulus extract showed strongest inhibitory potency with IC 50 value lower than 5 μg/mL. Meanwhile, pyrroloquinoline quinone (PQQ), widely used as a dietary supplement, was discovered as a promising PAD4 inhibitor with an IC 50 value lower than 4 μM. The inhibition kinetic analysis, drug affinity response target stability (DARTS) and molecular docking were performed to confirm the interaction between PQQ and PAD4. This method has great potential for researchers to monitor activities and discover potential inhibitors of PAD4., 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.)

Electrostatic adsorption is an enzyme immobilization method that effectively maintains enzyme activity and exhibits considerable binding efficiency. However, enzymes carry different charges at their respective reaction pH levels, which prevents the use of the same carrier to immobilize enzymes with different charges. In this study, we employed a template-mediated polysaccharide-enzyme coupling self-assembly strategy to develop a charge-controllable supramolecular immobilization carrier by regulating the charge properties of carboxymethyl chitosan, enabling the universal immobilization of enzymes with different charge levels across a range of reaction pH values. By using silica nanoparticles of certain sizes as templates, the size of the carrier can be precisely controlled and the hollow network structure formed after removing the template can effectively reduce mass transfer resistance. Trypsin and papain are used as model enzymes, and the experimental results show that the supramolecular self-assembly immobilization strategy does not disrupt the secondary structure of the enzyme molecules. After 2 h of reaction, the enzyme activities of immobilized papain and immobilized trypsin are 13.2% and 7.7% higher than those of the free enzymes, respectively. After 10 consecutive reactions, the enzyme activities of immobilized papain and immobilized trypsin retained 56.3% and 64.3% of their initial values, respectively.

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 effects of common migration substances in milk packaging on digestive protease were studied. We choose the common migrants found in eight types of multi-layer composite milk packaging. Enzyme activity experiments revealed that pepsin activity decreased by approximately 18 % at 500 μg/mL of stearic acid and stearamide treatment, while trypsin activity decreased by approximately 18 % only by stearic acid treatment (500 μg/mL). Subsequently, fluorescence spectroscopy, circular dichroism spectroscopy, and molecular docking technology were employed to investigate the inhibition mechanism of protease activity by migrating substances in three systems: stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin. Results showed that the inhibitory effect of stearic acid on trypsin is a reversible mixed inhibition, whereas the inhibitory effects of stearic acid and stearamide on pepsin are non-competitive. In all three systems, ΔH < 0, ΔS < 0, and ΔG < 0, indicating the binding process between the migrant and the protease is a spontaneous exothermic process primarily driven by hydrogen bonding and van der Waals forces. In addition, their binding constants are all around 10 4 L/moL, indicating that there are moderate binding affinities exist between migrants and proteases. The binding process results in the quenching of the protease's endogenous fluorescence and induces alterations in the enzyme's secondary structure. Synchronized fluorescence spectroscopy showed that stearic acid enhanced the hydrophobicity near the Tyr residue of trypsin. The molecular docking results indicated that the binding affinity of stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin was -22.51 kJ/mol, -12.35 kJ/mol, -19.28 kJ/mol respectively, which consistent with the trend in the enzyme activity results. This study can provide references for the selection of milk packaging materials and the use of processing additives, ensuring food health and safety., 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.)

A key parameter of any bottom-up proteomics mass spectrometry experiment is the identity of the enzyme that is used to digest proteins in the sample into peptides. The Casanovo de novo sequencing model was trained using data that was generated with trypsin digestion; consequently, the model prefers to predict peptides that end with the amino acids "K" or "R". This bias is desirable when Casanovo is used to analyze data that was also generated using trypsin but can be problematic if the data was generated using some other digestion enzyme. In this work, we modify Casanovo to take as input the identity of the digestion enzyme alongside each observed spectrum. We then train Casanovo with data generated by using several different enzymes, and we demonstrate that the resulting model successfully learns to capture enzyme-specific behavior. However, we find, surprisingly, that this new model does not yield a significant improvement in sequencing accuracy relative to a model trained without enzyme information but using the same training set. This observation may have important implications for future attempts to make use of experimental metadata in de novo sequencing models.

Apolipoprotein A-I (ApoA-I), one of the most abundant proteins in plasma and the major protein component of high-density lipoprotein (HDL), is naturally found in several proteoforms; two of them are ProApoA-I and mature ApoA-I. These two proteoforms of ApoA-I coexist in biological samples and differ only in their N-terminal end. Virtually, the only way to differentiate them is by detecting the proteoform-specific N-terminal proteolytic peptides (RHFWQQDEPPQSPWDR and DEPPQSPWDR, respectively) using liquid chromatography in multiple reaction monitoring mode mass spectrometry (LC-MRM-MS). We have developed a bottom-up LC-MRM-MS method to simultaneously detect proApoA-I and mature ApoA-I. To test the specificity of the method, we digested with trypsin purified mature ApoA-I and recombinant proApoA-I. As expected, only the N-term peptide corresponding to the mature ApoA-I proteoform (DEPPQSPWDR) was detected when digesting mature ApoA-I. However, the digestion of the proApoA-I produced not only the N-terminal peptide corresponding to proApoA-I (RHFWQQDEPPQSPWDR) but also the N-terminal tryptic peptide corresponding to mature ApoA-I (DEPPQSPWDR). This effect was produced by standard and high-specificity trypsin as well as by the Arg-C enzyme in a self-limited manner (approximately 10% of the total). The synthetic proApo-I peptide is not cleaved by trypsin, suggesting that the here reported effect is dependent on protein conformation. The effect is not negligible, as it can be detected by LC-MRM-MS, and correction calculations should be applied to accurately quantify proApoA-I and mature ApoA-I in biological samples where these two proteoforms may coexist.

Pancreatic lipase (PNLIP) is the major lipolytic enzyme secreted by the pancreas. A recent study identified human PNLIP variants P245A, I265R, F300L, S304F, and F314L in European cohorts with chronic pancreatitis. Functional analyses indicated that the variants were normally secreted but exhibited reduced stability when exposed to pancreatic proteases. Proteolysis of the PNLIP variants yielded an intact C-terminal domain, while the N-terminal domain was degraded. The protease-sensitive PNLIP phenotype was strongly correlated with chronic pancreatitis, suggesting a novel pathological pathway underlying the disease. To facilitate preclinical mouse modeling, here we investigated how the human mutations affected the secretion and proteolytic stability of mouse PNLIP. We found that variants I265R, F300L, S304F, and F314L were secreted at high levels, while P245A had a secretion defect and accumulated inside the cells. Proteolysis experiments indicated that wild-type mouse PNLIP was resistant to cleavage, while variant I265R was readily degraded by mouse trypsin and chymotrypsin C. Variants F300L, S304F, and F314L were unaffected by trypsin but were slowly proteolyzed by chymotrypsin C. The proteases degraded the N-terminal domain of variant I265R, leaving the C-terminal domain intact. Structural analyses suggested that changes in stabilizing interactions around the I265R mutation site contribute to the increased proteolytic susceptibility of this variant. The results demonstrate that variant I265R is the best candidate for modeling the protease-sensitive PNLIP phenotype in mice., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Universal sample preparation for proteomic analysis that enables unbiased protein manipulation, flexible reagent use, and low protein loss is required to ensure the highest sensitivity of downstream liquid chromatography-mass spectrometry (LC-MS) analysis. To address these needs, we developed a ZnCl 2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl 2 and methanol-induced protein precipitation at room temperature (RT). ZASP can remove trypsin digestion and LC-MS incompatible detergents such as SDS, Triton X-100, and urea at high concentrations in solution and unbiasedly recover proteins independent of the amount of protein input. We demonstrated the sensitivity and reproducibility of ZASP in an analysis of samples with 1 μg to 1000 μg of proteins. Compared to commonly used sample preparation methods such as SDC-based in-solution digestion, acetone precipitation, FASP, and SP3, ZASP has proven to be an efficient approach. Here, we present ZASP, a practical, robust, and cost-effective proteomic sample preparation method that can be applied to profile different types of samples., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteases play a crucial role in industrial enzyme formulations, with activity fluctuations significantly impacting product quality and yield. Therefore, developing a method for precise and rapid detection of protease activity is paramount. This study aimed to develop a rapid and accurate method for quantifying trypsin activity using integrated infrared (IR) and ultraviolet (UV) spectroscopy combined with data fusion techniques. The developed method evaluates the enzymatic activity of trypsin under varying conditions, including temperature, pH, and ionic strength. By comparing different data fusion methods, the study identifies the optimal model for accurate enzyme activity prediction. The results demonstrated significant improvements in predictive performance using the feature-level data fusion approach. Additionally, substituting the spectral data of the samples in the validation sets into the best prediction model resulted in a minimal residual difference between predicted and true values, further verifying the model's accuracy and reliability. This innovative approach offers a practical solution for the efficient and precise quantification of enzyme activity, with broad applications in industrial processes., 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.)

Immunoglobulin G (IgG) exhibits potent antiviral, antibacterial, and immunological activities. The digestion process and bioavailability of IgG are often a concern. Dietary hydrocolloids are crucial for regulating healthy digestion and the bioavailability of protein as functional components. Understanding the effects of dietary hydrocolloids on the digestive kinetics of IgG is requisite. Herein, the pepsin and trypsin digestion of IgG was investigated using ordered porous layer interferometry (OPLI). The real-time variation in the interference spectral shift reflected by OPLI can be converted into changes in the optical thickness (OT) to obtain a degradation kinetics curve. The impact of dietary hydrocolloids, including alginic acid sodium salt (ALG), polydextrose (PD), and konjac glucomannan (KG), on IgG degradation was evaluated using OPLI. The results demonstrated that ALG significantly inhibited the degradation of IgG by pepsin under acidic conditions, whereas the addition of PD increased the Michaelis-Menten constant for IgG degradation by trypsin. Notably, this dependence is not based on the hydrocolloid viscosity, but relies more on the electrical properties. The study enhances our understanding of how hydrocolloids affect IgG digestion and could provide valuable insights into preserving IgG activity and facilitating the development of oral drugs or health products related to IgG., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Complex and stubborn bacterial biofilm infections significantly hinder diabetic wound healing and threaten public health. Therefore, a dressing material that effectively clears biofilms and promotes wound healing is urgently required. Herein, we introduce a novel strategy for simultaneously dispersing extracellular polymeric substances and eradicating drug-resistant bacteria. We prepared an ultrabroad-spectrum and injectable quaternized chitosan (QCS) hydrogel loaded with trypsin, which degrades biofilm extracellular proteins. Increased temperature initiated QCS gelation to form the hydrogel, enabling the sustained release of trypsin and effective adherence of the hydrogel to irregularly shaped wounds. To reproduce clinical scenarios, biofilms formed by a mixture of Staphylococcus aureus (S. aureus), Methicillin-resistant S. aureus, and Pseudomonas aeruginosa were administered to the wounds of rats with streptozotocin-induced diabetes. Under these severe infection conditions, the hydrogel efficiently suppressed inflammation, promoted angiogenesis, and enhanced collagen deposition, resulting in accelerated healing of diabetic wounds. Notably, the hydrogel demonstrates excellent biocompatibility without cytotoxicity. In summary, we present a trypsin-loaded QCS hydrogel with tremendous clinical applications potential for the treatment of chronic infected wounds., 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.)

Though the heparin-protamine complex (HP complex) is a crucial system utilized in clinical settings, the metabolic pathways of this complex remain inadequately understood. Herein, the enzymatic degradation of the heparin-protamine complex by trypsin and its broader implications were investigated. By utilizing fluorescent gold nanoclusters liganded with the HP complex (AuNCs-HP complex), we observed significant morphological and spectral changes during enzymatic degradation. Experiments showed that AuNCs-HP complex could be degraded and cleaved into small fragments by trypsin. Moreover, the AuNCs-HP complex demonstrated its potential as a highly sensitive spectral sensing platform, enabling precise measurement of trypsin activity with an outstanding detection limit (0.34 ng mL -1 ). Additionally, we explored its utility for specific tumor cell detection, focusing on lung adenocarcinoma cells, and successfully identified their presence through distinctive fluorescence changes. These remarkable findings not only contribute valuable insights into targeted degradation systems but also offer promising opportunities for cancer biomarker detection. The AuNCs-HP complex serves as an innovative tool for real-time trypsin activity monitoring, paving the way for advanced biomedical applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Backgrounds: Various physiological functions and reaction cascades, as well as disease progression in the living systems, are controlled by the activity of specific proteolytic enzymes. We conducted the study to evaluate protease activity by assessing peptide fragments from either conserved or labeled red blood cells (RBCs) with aminofluorescein (AF) in the reaction media. Methods: RBCs were incubated in media containing trypsin. Subsequently, the concentration of peptide fragments in the reaction media, resulted by the digestion with trypsin from conserved cells, was estimated by 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) as an amine-reactive fluorogenic reagent. In a second approach, we conjugated AF to the conserved RBCs and then exposed AF-labeled RBCs to trypsin. This was followed by directly measuring the fluorescence intensity (FI) in the reaction media to estimate the concentration of AF-labeled peptide fragments resulting from the enzyme's activity. Results: Show a concentration- and time-dependent increase in FIs, reflecting the activity of trypsin as a proteolytic enzyme. The FIs increased significantly by 4 to 5 folds in samples treated with different enzyme concentrations, and by over 11 folds after 2 h incubation in media containing a 50  μ L trypsin, as evidenced by CBQCA assays. Conclusion: These fast and affordable approaches could be applied with high reliability for the general estimation of protease activity in samples and customized for diagnostic purposes and prognostic evaluation in various diseases., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Mohamed K. Al-Essa et al.)

The accurate detection of Protamine and Trypsin, two biomolecules with significant clinical and biological relevance, presents a substantial challenge because of their structural peculiarities, low abundance in physiological fluids, and potential interference from other substances. Protamine, a cationic protein, is crucial for counteracting heparin overdoses, whereas Trypsin, a serine protease, is integral to protein digestion and enzyme activation. This study introduces a novel fluorescence sensor based on a (4-(1,2,2-tris(4-phosphonophenyl)vinyl)phenyl)phosphonic acid octasodium salt (TPPE), leveraging aggregation-induced emission (AIE) characteristics and electrostatic interactions to achieve selective and sensitive detection of these biomolecules. Through comprehensive optical characterization, including ground-state absorption, steady-state, and time-resolved emission spectroscopy, the interaction mechanisms and aggregation dynamics of TPPE with Protamine and Trypsin were elucidated. The sensor exhibits very high sensitivity (LOD: 1.45 nM for Protamine and 32 pM for Trypsin), selectivity, and stability, successfully operating in complex biological matrices, such as human serum and urine. Importantly, this sensor design underscores the synergy between the AIE phenomena and biomolecular interactions, offering a promising alternative for analytical applications in biomedical research and clinical diagnostics. The principles outlined herein open new avenues for the development of other AIE-based sensors, expanding the toolkit available for detecting a wide range of biomolecules using similar design strategies.

In this study, a hydroxyl radical oxidation system was established to simulate the oxidation process in fermented meat products. This system was employed to examine the structural changes in myofibrillar proteins (MPs) resulting from tryptic hydrolysis after a hydroxyl radical oxidative regime. The effect of these changes on the ability of MPs to bind selected aldehydes (3-methyl butanal, pentanal, hexanal, and heptanal) was also investigated. Moderate oxidation (H 2 O 2  ≤ 1.0 mM) unfolded the structure of MPs, facilitating trypsin-mediated hydrolysis and increasing their binding capacity for the four selected aldehydes. However, excessive oxidation (H 2 O 2  ≥ 2.5 mM) led to cross-linking and aggregation of MPs, inhibiting trypsin-mediated hydrolysis. The oxidised MPs had the best binding capacity for heptanal. The interaction of the oxidised trypsin-hydrolysed MPs with heptanal was driven by hydrophobic interactions. The binding of heptanal affected the structure of the oxidised trypsin-hydrolysed MPs and reduced their α-helix content., 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 © 2023. Published by Elsevier Ltd.)

Hyperpolarization derived from water protons enhances the NMR signal of 15 N nuclei in a small molecule, enabling the sensitive detection of a protein-ligand interaction. The water hyperpolarized by dissolution dynamic nuclear polarization (D-DNP) acts as a universal signal enhancement agent. The 15 N signal of benzamidine was increased by 1480-fold through continuous polarization transfer by J -coupling-mediated cross-polarization ( J -CP) via the exchangeable protons. The signal enhancement factor favorably compares to factors of 110- or 17-fold using non-CP-based polarization transfer mechanisms. The hyperpolarization enabled detection of the binding of benzamidine to the target protein trypsin with a single-scan measurement of 15 N R 2 relaxation. J -CP provides an efficient polarization mechanism for 15 N or other low-frequency nuclei near an exchangeable proton. The hyperpolarization transfer sustained within the relaxation time limit of water protons additionally can be applied for the study of macromolecular structure and biological processes.

Background: Bile's potential to reflect the health of the biliary system has led to increased attention, with proteomic analysis offering deeper understanding of biliary diseases and potential biomarkers. With the emergence of normothermic machine perfusion (NMP), bile can be easily collected and analyzed. However, the composition of bile can make the application of proteomics challenging. This study systematically evaluated various trypsin digestion methods to optimize proteomics of bile from human NMP livers., Methods: Bile was collected from 12 human donor livers that were accepted for transplantation after the NMP viability assessment. We performed tryptic digestion using six different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without additional precipitation before digestion. Proteins were analyzed using untargeted proteomics. Methods were assessed for total protein IDs, variation, and protein characteristics to determine the most optimal method., Results: Methods involving precipitation surpassed crude methods in protein identifications (4500 vs 3815) except for in-gel digestion. Filtered data (40%) resulted in 3192 versus 2469 for precipitated and crude methods, respectively. We found minimal differences in mass, cellular components, or hydrophobicity of proteins between methods. Intermethod variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming others. Age-related biological comparisons revealed upregulation of metabolic-related processes in younger donors and immune response and cell cycle-related processes in older donors., Conclusions: Variability between methods emphasizes the importance of cross-validation across multiple analytical approaches to ensure robust analysis. We recommend the in-gel crude method for its simplicity and efficiency, avoiding additional precipitation steps. Sample processing speed, cost, cleanliness, and reproducibility should be considered when a digestion method is selected for bile proteomics.

The presence of Ca 2+ ions is known to facilitate the activity of trypsin-like serine proteases via structural stabilization against thermal denaturation and autolysis. Herein, we report a new and hidden regulatory role of Ca 2+ in the catalytic pathways of trypsin and α-chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of trypsin via liquid-liquid phase separation to yield membraneless condensates over a broad range of concentrations, pH, and temperature, which are stabilized by multivalent hydrophobic interactions. Interestingly, we found that Ca 2+ binding in the calcium binding loop reversibly regulates the condensation of trypsin and α-chymotrypsin. Spontaneous condensation effectively prevents autolysis of trypsin and preserves its native-like esterase activity for a prolonged period of time. It has also been found that phase-separated trypsin responds to Ca 2+ -dependent activation of its esterase activity even after 14 days of storage while free trypsin failed to do so. The present study highlights an important physiological aspect by which cells can spatiotemporally regulate the biocatalytic efficacy of trypsin-like serine proteases via Ca 2+ -signaling.

Metal-organic frameworks and hydrogen-organic frameworks (MOFs and HOFs) are attractive hosts for enzyme immobilization, but they are limited to immobilizing the purified enzymes, making industrial upscaling unattractive. Herein, aptamer-modified dual thermoresponsive polymeric micelles with switchable self-assembly and core-shell structure are constructed, which enable selective immobilization of trypsin directly from complex biological systems through a cascade operation of separation and immobilization. Their steric self-assembly provides a large amount of adsorption sites on the soluble micellar shell, resulting in high adsorption capacity and excellent selectivity. Meanwhile, their aptamer affinity ligand and cavity maintain the native conformations of trypsin and offer protective effects even in harsh conditions. The maximum adsorption capacity of the polymeric micelles for trypsin was determined to be 197 mg/g at 60 min, superior to those of MOFs and HOFs. 67.2 and 86.6% of its original activity was retained for trypsin immobilized in the cavity under strong alkaline and acidic conditions, respectively.

A novel carbazole compound, named 1-(9-ethyl-9H-carbazol-3-yl)-3-phenylurea (Cpu) was synthesized and its binding properties with protease enzymes (pepsin and trypsin) has been examined by steady-state fluorescence measurements, UV/vis absorption, infrared (FT-IR) and circular dicroism (CD) spectroscopies and also computational methods. The fluorescence experimental results indicated that the quenching mechanism of enzyme by Cpu is static process. The thermodynamic parameters (both negative ΔH/ΔS) and molecular docking results suggested that the binding of Cpu to pepsin/trypsin were driven by hydrogen bonds and van der Waals forces. Based on Förster's theory, the binding distance (r) between pepsin/trypsin and Cpu was calculated to be 3.072/2.784 nm, which implies that non-radiative energy transfer occurs from enzyme to Cpu. Furthermore, absorption, CD, and FT-IR spectral analysis provided an evidence that the presence of Cpu induced notable changes in the secondary structures and microenvironmental of both pepsin and trypsin, supporting its significant influence on these enzymes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The detection of trypsin and its inhibitors is important for both clinical diagnosis and disease treatment. Abnormal trypsin activity affects pancreatic function and leads to corresponding pathological changes in the body. Therefore, the study presented a riboflavin-induced photo-ATRP electrochemical assay of trypsin activity and its inhibitor, including detection of trypsin activity in real urine samples. Experiments were performed on indium tin oxide (ITO) electrodes modified with sulfhydryl groups of 3-mercaptopropionic acid, and target trypsin-specific cleavage of BSA-Au nanocluster (BSA-Au NCs) was followed by the modification of Au NCs to the electrodes using Au-S. The Au NCs immobilized monodeoxy-monomercapto-β-cyclodextrin@adamantan-2-amine (SH-β-CD@2-NH 2 -Ada) host-guest inclusion complexes to the electrode surfaces via Au-S. In a two-component photo-initiator system consisting of riboflavin as an initiator and ascorbic acid (AA) as a mild reducing agent under mild blue light radiation, a large number of electroactive substances were grafted onto the electrode surface to generate electrochemical signals. In addition, we have successfully realized the detection of clinical drug inhibitors of trypsin. The detection limit of the system is as low as 0.0024 ng/mL, which much littler than the average standard of trypsin in the patient's urine or serum. It's worth noting that this work will provide researchers with a different route to design electrochemical sensors based on non-covalent recognition strategies., 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.)

Heparin is a highly negatively charged sulfated linear polymer glycosaminoglycan that has been widely used as an anticoagulant in medicine. Protamine is a cationic protein rich in arginine that is used to treat the blood-brain barrier during excess heparin surgery. Trypsin is the most important digestive enzyme-encoding generated by the pancreas and can specifically cleave the carboxyl ends of arginine and lysine residues. Heparin, protamine, and trypsin interact and constrain each other, and their fluctuations reflect the body's dysfunction. Therefore, it is necessary to develop a fast, sensitive, and highly selective assay for regularly monitoring the levels of heparin, protamine, and trypsin in serum. Herein, a fluorescent and colorimetric dual-mode upconversion nanoparticle (UCNP) biosensor was used for the determination of heparin, protamine, and trypsin based on the oxidase-mimicking activity of Ce 4+ and electrostatic control. The biosensor exhibited sensitive detection of heparin, protamine, and trypsin with low limits of detection (LODs) of 16 ng/mL, 87 ng/mL and 31 ng/mL, respectively. Furthermore, the designed biosensor could eliminate autofluorescence, which not only effectively increased the accuracy of the sensor but also provided a new sensing pathway for the detection of differently charged biotargets., 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.)

Modification of catalytic expression of enzymes and regulating their in vivo activity are the goals of novel treatment strategies. A green synthetic nanostructured silver with potent trypsin inhibitory properties has not yet been developed, despite the fact that silver nanoparticles possess unique properties that allow them to efficiently block enzymes. The present study demonstrates for the first time a facile, safe, economic, and eco-friendly synthetic route for silver nanoparticles using an aqueous extract of Holigarna arnottiana bark engineered to interact with trypsin and hinder its activity effectively. The studies carried out to examine the interaction between these biofabricated AgNPs (HaAgNPs) and trypsin by UV-visible spectrophotometry and FTIR spectroscopy suggest that the formation of trypsin-HaAgNP complex is responsible for diminishing the catalytic efficiency of trypsin. In vivo studies on Aedes aegypti larval serum support these instrumental results of HaAgNP-induced trypsin inhibition and proves its application as a biopesticide. It is noteworthy that the bioengineered HaAgNPs were also found to have good inhibition potential against pepsin and urease as well. A variety of methods have been employed to characterize the synthesized biocompatible HaAgNPs and it possesses a characteristic absorption maximum of 420 nm. Their shelf life of above 7 years is noticeable, since none of the reported green synthesized AgNPs possess a shelf life of more than 1 year. Altogether, this work demonstrates that biofabricated HaAgNPs are multifunctional and cost-resilient biological tools that can be used as enzyme regulators possessing antioxidant, antimicrobial, and insecticidal features., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, the interaction mechanism and native conformational variation of trypsin (Try) affected by CeO 2 nanoparticles (NPs) were systematically studied via various spectroscopic methods. The results of fluorescence spectroscopy revealed that CeO 2 NPs markedly quenched the endogenous fluorescence of Try via the mechanism of static quenching. The main forces that contributed to the binding of Try and CeO 2 NPs were van der Waals forces, hydrogen bonds, and electrostatic forces, as observed by the binding constants and significant thermodynamic characteristics of the two substances. The incorporation of CeO 2 NPs lead to a slight change in the structure of Try, as shown by synchronized fluorescence spectroscopy, three-dimensional fluorescence spectroscopy and circular dichroism (CD) spectroscopy. Moreover, the enzyme activity of Try decreased with the addition of CeO 2 NPs. This study is highly important for fully evaluating the use of CeO 2 NPs in biomedical sciences and is helpful for clarifying the mechanism between Try and CeO 2 NPs at the molecular level., 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.)

The interfacial behavior of soy hull polysaccharide (SHP) at the oil-water interface and the stabilization mechanism of high internal phase emulsion (HIPE) with three enzymes (α-amylase, trypsin and papain) were investigated. The diameter of the α-amylase-treated emulsion was the minimum at 40 min, indicating that the carbohydrate portions of SHP form a thick layer on the surface of the droplet to prevent aggregation. Moreover, Raman spectroscopy revealed significantly higher levels of disordered content of SHP emulsion treated with α-amylase at 60 min, potentially affecting the directional movement of SHP molecules in the emulsion. Conversely, the content of β-sheet and β-turn was lower than trypsin and papain, possibly due to ion-dipole interaction between the polar group residues within SHP and ions, or protonation with H + ., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024. Published by Elsevier Ltd.)

Multimodal biosensors with independent signaling pathways can self-calibrate and improve the reliability of disease biomarker detection. Herein, a colorimetric-fluorescent dual-mode paper-based biosensor with PAN/Fe(III)-CNOs (FPCs) as core components has been developed, which information is recognized by smartphone and naked eye. Using 1-(2-pyridylazo)-2-naphthol (PAN) as a mediator, Fe(III) is enriched on the surface of carbon nano-onions (CNOs), endowing FPCs with excellent mimetic enzyme activity and photothermal conversion ability, which allows it to output amplified colorimetric signals under laser irradiation. In addition, the complexation of PAN with Fe(III) broadens its absorption spectrum, which makes FPCs more suitable to be energy acceptors to quench fluorescence of polymer dots (Pdots), resulting in the changes of output fluorescent signal. Based on the above design, a portable colorimetric-fluorescent dual-mode biosensor is proposed for trypsin detection with Pdots as fluorescence sources and FPCs as fluorescence quenchers and nanoenzymes. This work provides a convenient way for constructing portable visual multimodal biosensors, which is expected to applied in various disease diagnosis., 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.)

A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and K m value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30-50 °C and pH range of 4.0-10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The mechanisms of trypsin hydrolysis time on the structure of soy protein hydrolysate fibril aggregates (SPHFAs) and the stability of SPHFAs-high internal phase Pickering emulsions (HIPPEs) were investigated. SPHFAs were prepared using soy protein hydrolysate (SPH) with different trypsin hydrolysis time (0 min-120 min) to stabilize SPHFAs-HIPPEs. The results showed that moderate trypsin hydrolysis (30 min, hydrolysis degree of 2.31 %) induced SPH unfolding and increased the surface hydrophobicity of SPH, thereby promoting the formation of flexible SPHFAs with maximal thioflavin T intensity and ζ-potential. Moreover, moderate trypsin hydrolysis improved the viscoelasticity of SPHFAs-HIPPEs, and SPHFAs-HIPPEs remained stable after storage at 25 °C for 80 d and heating at 100 °C for 1 h. Excessive trypsin hydrolysis (> 30 min) decreased the stability of SPHFAs-HIPPEs. In conclusion, moderate trypsin hydrolysis promoted the formation of flexible SPHFAs with high surface charge by inducing SPH unfolding, thereby promoting the stability of SPHFAs-HIPPEs., 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: As anticoagulants are widely used to treat patients with atrial fibrillation (AF) and other thrombotic conditions, it is necessary for physicians to determine whether the medication has been taken in emergencies. Among many anticoagulants, rivaroxaban has attracted attention due to its safety and convenience. Since rivaroxaban inhibits activated coagulation factor X (factor Xa), measuring factor Xa activity can determine the presence of rivaroxaban., Results: We report a magnetic biosensing platform capable of measuring the activity of factor Xa using peptide substrates conjugated with magnetic nanoparticles (MNPs). Due to the size of factor Xa, a new method of solution-phase assays was proposed for magnetic biosensing platforms to address issues with immobilized peptides on the sensors. This method was validated with factor Xa and trypsin, both of which are serine proteases. In the solution-phase assays, samples with either the enzymes of interest or no enzyme were simultaneously measured, and the activity of the enzyme was estimated using the difference between the measurements. Unlike conventional optical methods, our platform was capable of detecting the activity of factor Xa at 2 μg mL -1 with a 30 min sample incubation., Significance: The assay using giant magnetoresistive biosensors outperformed conventional optical techniques. This platform can facilitate the determination of the presence of rivaroxaban and assist physicians in deciding on appropriate treatments for patients., 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.)

Chilling injury (CI) in green pepper fruits during low-temperature storage causes a significant decline in quality. The present study utilized physiological, transcriptomic, and metabolomic analyses to idneitfy the mechanisms by which trypsin mitigates CI in green peppers stored at 4 °C for 8 days, followed by 3 days of shelf life. Results indicated that the trypsin treatment significantly reduced electrolyte leakage and the CI index in peppers, effectively extending their shelf life and preserving postharvest quality. After 4 days of storage, comparative -omic analyses identified 2514 differentially expressed genes (DEGs) and 397 differentially abundant metabolites (DAMs) between trypsin-treated and control peppers. The trypsin treatment induced changes in sugar metabolism, modulating the expression of HK, SUS, INV, and GLGC, which affected the abundance of metabolites such as CDP-glucose and α-D-p-glucose. Trypsin also enhanced carotenoid metabolism, altering the abundance of rhodopinal glucoside, 1'-hydroxyl-γ-carotene glucoside, and farnesyl 1-PP, and influencing the expression of PDS, CRTH, CRTB, and LUT5. Notably, the trypsin treatment activated the mitogen-activated protein kinase (MAPK) pathway that plays an integral role in the signal transduction of abiotic stress. Differential expression of FLS2, ELF18, PTO, PR1, PTI5, WPKY, MEKK1, and MPK6 genes in the MAPK pathway was observed, which was correlated with CI mitigation in green peppers during cold storage. In conclusion, trypsin is an effective treatment for reducing CI in green peppers during cold storage. The present study provides valuable insights into its physiological and molecular impact on green pepper fruit., 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 Ltd.)

The effective method for trypsin purification should be established because trypsin has important economic value. In this work, a novel and simple strategy was proposed for fabricating micron-sized magnetic Fe 3 O 4 @agarose-benzamidine beads (MABB) with benzamidine as a ligand, which can efficiently and selectively capture trypsin. The micro-sized MABB, with clear spherical core-shell structure and average particle size of 6.6 μm, showed excellent suspension ability and magnetic responsiveness in aqueous solution. The adsorption capacity and selectivity of MABB towards target trypsin were significantly better than those of non-target lysozyme. According to the Langmuir equation, the maximum adsorption capacity of MABB for trypsin was 1946 mg g -1  at 25 °C, and the adsorption should be a physical sorption process. Furthermore, the initial adsorption rate and half equilibrium time of MABB toward trypsin were 787.4 mg g -1  min -1 and 0.71 min, respectively. To prove the practicability, MABB-based magnetic solid-phase extraction (MSPE) was proposed, and the related parameters were optimized in detail to improve the purification efficiency. With Tris-HCl buffer (50 mM, 10 mM CaCl 2 , pH 8.0) as extraction buffer, Tris-HCl buffer (50 mM, 100 mM CaCl 2 , pH 8.0) as rinsing buffer, acidic eluent (0.01 M HCl, 0.5 M NaCl, pH 2.0) as eluent buffer and alkaline buffer (1 M Tris-HCl buffer, pH 10.0) as neutralization solution, the MABB-based MSPE was successfully used for trypsin purification from the viscera of grass carp (Ctenopharyngodon idella). The molecular weight of purified trypsin was determined as approximate 23 kDa through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified trypsin was highly active from 30 °C to 60 °C, with an optimum temperature of 50 °C, and was tolerant to pH variation, exhibiting 85 % of maximum enzyme activity from pH 7.0 to 10.0. The results demonstrated that the proposed MABB-based MSPE could effectively purify trypsin and ensure the biological activity of purified trypsin. Therefore, we believe that the novel MABB could be applicable for efficient purification of trypsin from complex biological systems., 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.)

Creating molecules capable of inhibiting ice recrystallization is an active research area aiming to improve the freeze-thaw characteristics of foods and biomedical materials. Peptide mixtures have shown promise in preventing freezing-induced damage, but less is known about the relationship between their amino acid compositions and ice recrystallization inhibition (IRI) activities. In this article, we used Ni 2+ immobilized metal affinity chromatography (IMAC) to fractionate pulse protein hydrolysates, created by Alcalase and trypsin, into mixtures lacking and enriched in His, and Cys residues. The aim of this study was to fractionate pulse protein hydrolysates based on their amino acid compositions and evaluate their resulting physicochemical and IRI characteristics. Ni 2+ IMAC fractionation induced IRI activity in all of the evaluated soy, chickpea, and pea protein hydrolysates regardless of their amino acid composition. Ni 2+ IMAC fractionation produced chemically distinct fractions of peptides, differing by their molecular weights, amino acid composition, and IRI activities. The resulting peptide mixtures' molecular weight, amino acid composition, secondary structure, and sodium ion levels were found to have no correlation with their IRI activities. Thus, we demonstrate for the first time the ability of Ni 2+ IMAC fractionation to induce IRI activity in hydrolyzed pulse proteins., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2024. Published by Elsevier Ltd.)

Aeruginosins are common metabolites of cyanobacteria. In the course of re-isolation of the known aeruginosins KT608A and KT608B for bioassay studies, we isolated three new sulfated aeruginosins, named aeruginosins KT688 ( 1 ), KT718 ( 2 ), and KT575 ( 3 ), from the extract of a Microcystis cell mass collected during the 2016 spring bloom event in Lake Kinneret, Israel. The structures of the new compounds were established on the basis of analyses of the 1D and 2D NMR, as well as HRESIMS data. Marfey's method, coupled with HR ESI LCMS and chiral HPLC, was used to establish the absolute configuration of the amino acid and hydroxyphenyl lactic acid residues, respectively. Compounds 1 - 3 were tested for inhibition of the serine protease trypsin, and compounds 1 and 2 were found to exhibit IC 50 values of 2.38 and 1.43 µM, respectively.

A variety of therapeutic possibilities have emerged for skillfully regulating protein function or conformation through intermolecular interaction modulation to rectify abnormal biochemical reactions in diseases. Herein, a devised strategy of enzyme coordinators has been employed to alleviate postoperative pancreatic fistula (POPF), which is characterized by the leakage of digestive enzymes including trypsin, chymotrypsin, and lipase. The development of a dextrorotary (D)-peptide supramolecular gel (CP-CNDS) under this notion showcases its propensity for forming gels driven by intermolecular interaction. Upon POPF, CP-CNDS not only captures enzymes from solution into hydrogel, but also effectively entraps them within the internal gel, preventing their exchange with counterparts in the external milieu. As a result, CP-CNDS completely suppresses the activity of digestive enzymes, effectively alleviating POPF. Remarkably, rats with POPF treated with CP-CNDS not only survived but also made a recovery within a mere 3-day period, while mock-treated POPF rats had a survival rate of less than 5 days when experiencing postoperative pancreatic fistula, leak or abscess. Collectively, the reported CP-CNDS provides promising avenues for preventing and treating POPF, while exemplifying precision medicine-guided regulation of protein activity that effectively targets specific pathogenic molecules across multiple diseases., (© 2024. The Author(s).)

An electrophilic arginine mimetic, 2-chloroacetamidine (CAM), was deployed to enable trypsin-mediated proteolysis at cysteine residues and to enhance mass spectrometry-based proteomic detection of cysteine-containing peptides. Illustrating the value of the CAM-capping strategy, proteogenomic analysis using a two-stage false discovery rate (FDR) search revealed >50% enhanced coverage of missense variants, when compared to established workflows.

The development of a green and facile method for the controlled synthesis of functional polypeptides is desired for sustainable material applications. In this study, the regioselective synthesis of poly(l-lysine) (polyLys) via enzyme-catalyzed aminolysis was achieved by bulk polymerization of l-lysine ethyl ester (Lys-OEt) using immobilized Candida antarctica lipase Novozym 435 (IM-lipase) or trypsin (IM-trypsin). Structural characterization of the obtained polyLys revealed that IM-lipase resulted solely in ε-linked amide bond formation, whereas IM-trypsin predominantly provided α-linked polyLys. Optimization of the conditions for the bulk polymerization using immobilized enzymes resulted in high monomer conversion and a high degree of polymerization, with excellent regioselectivity. Molecular docking simulations revealed different binding conformations of Lys-OEt to the catalytic pockets of lipase and trypsin, which putatively resulted in different amino moieties being used for amide bond formation. The immobilized enzymes were recovered and recycled for bulk polymerization, and the initial activity was maintained in the case of IM-trypsin. The obtained α- and ε-linked polyLys products exhibited different degradability against proteolysis, demonstrating the possibility of versatile applications as sustainable materials. This enzymatic regioregular control enabled the synthesis of well-defined polypeptide-based materials with a diverging structural variety.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18 O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

This study aimed to fabricate gum Arabic (GA)-coated Fe 3 O 4 nanoparticles bearing numerous active aldehyde groups on their surface, followed by an assessment of their capability as a magnetic support for the covalent immobilization of the trypsin enzyme for the first time. FT-IR, XRD, TGA, and SEM results demonstrated the successful synthesis of GA-coated Fe 3 O 4 nanoparticles, along with the covalent immobilization of the enzyme onto the support. Immobilization enhanced the relative enzymatic activity across a range of aqueous solution pH levels (ranging from 4 to 11) and temperatures (ranging from 20 to 80 °C) without altering the optimum pH and temperature for trypsin activity. Kinetic studies using Michaelis-Menten plots revealed changes in kinetic parameters, including a lower V max and higher K m for immobilized trypsin compared to the free enzyme. The immobilization onto magnetic gum Arabic nanoparticles resulted in an improved stability of trypsin in the presence of various solvents, maintaining a stability order comparable to that of the free enzyme due to the stabilizing effect of the support. The reusability results showed that the immobilized enzyme can retain over 93% of its activity for up to 15 cycles., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)