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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.)

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.

The Aedes aegypti mosquito plays a critical role in the transmission of viral diseases, including Zika virus, which poses significant public health challenges. Understanding the complex interactions between mosquitoes and viruses is paramount for the development of effective control strategies. In this study, we demonstrate that silencing the A. aegypti adiponectin receptor-like protein (AaARLP) results in a reduction of Zika virus infection. Transcriptomic analysis identified alterations in several trypsin genes and further revealed that AaARLP -knockdown mosquitoes had diminished trypsin activity. Moreover, silencing of selected trypsins resulted in a similar delay in Zika virus infection in mosquitoes, further highlighting the connection between the AaARLP and trypsin. Overall, our findings demonstrate that AaARLP signaling is important for Zika virus infection of A. aegypti ., Importance: Arboviruses pose a significant threat to public health, with mosquitoes, especially Aedes aegypti , being a major vector for their transmission. Gaining insight into the complex interaction between mosquitoes and viruses is essential to build successful control strategies. In this study, we identified a novel pathway connecting the A. aegypti adiponectin receptor-like protein and its association with trypsin, key enzymes involved in blood digestion. Furthermore, we demonstrated the significance of signaling via the adiponectin receptor-like protein in virus infection within the mosquito. Together, our discoveries illuminate mosquito metabolic pathways essential in viral infection., 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.)

Pancreatitis is a common, life-threatening inflammatory disease of the exocrine pancreas. Its pathogenesis remains obscure, and no specific or effective treatment is available. Gallstones and alcohol excess are major etiologies of pancreatitis; in a small portion of patients the disease is hereditary. Pancreatitis is believed to be initiated by injured acinar cells (the main exocrine pancreas cell type), leading to parenchymal necrosis and local and systemic inflammation. The primary function of these cells is to produce, store, and secrete a variety of enzymes that break down all categories of nutrients. Most digestive enzymes, including all proteases, are secreted by acinar cells as inactive proforms (zymogens) and in physiological conditions are only activated when reaching the intestine. The generation of trypsin from inactive trypsinogen in the intestine plays a critical role in physiological activation of other zymogens. It was proposed that pancreatitis results from proteolytic autodigestion of the gland, mediated by premature/inappropriate trypsinogen activation within acinar cells. The intra-acinar trypsinogen activation is observed in experimental models of acute and chronic pancreatitis, and in human disease. On the basis of these observations, it has been considered the central pathogenic mechanism of pancreatitis - a concept with a century-old history. This review summarizes the data on trypsinogen activation in experimental and genetic rodent models of pancreatitis, particularly the more recent genetically engineered mouse models that mimic mutations associated with hereditary pancreatitis; analyzes the mechanisms mediating trypsinogen activation and protecting the pancreas against its' damaging effects; discusses the gaps in our knowledge, potential therapeutic approaches, and directions for future research. We conclude that trypsin is not the culprit in the disease pathogenesis but, at most, a mediator of some pancreatitis responses. Therefore, the search for effective therapies should focus on approaches to prevent or normalize other intra-acinar pathologic processes, such as defective autophagy leading to parenchymal cell death and unrelenting inflammation., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

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.

Rice is commonly utilized as a wheat bread substitute due to its low allergenicity. However, rice bread faces challenges in processing efficiency and the formation of a cohesive gel network structure, resulting in suboptimal taste Hence, this study compared four improvers-trypsin, whey protein (WPC), hydroxypropyl methyl cellulose (HPMC), and molecularly distilled monoglycerides (GMSs). The impacts of the four improvers on the processing attributes of rice dough were comprehensively assessed across fermentation, moisture content analysis, rheology, heat stability, and pasting characteristics. The findings indicated that the incorporation of trypsin, HPMC, and WPC resulted in 107%, 61%, and 1% increases in gas production of fermented rice dough, respectively, while reducing the regrowth values to 564.00 ± 7.21, 176.67 ± 0.58, and 611.00 ± 3.61 cP. Notably, the air-holding capacity of HPMC-fermented rice dough exhibited a 7% enhancement. All four types of improvers raised the enthalpy of melting (ΔH) and the difference in melting point (ΔT) of fermented rice doughs, with trypsin enhancing ΔH by 44% and ΔT by 40%. GMS, HPMC, and WPC increased the degree of water incorporation in fermented doughs. This study could serve as a benchmark for enhancing the fermentation attributes of rice dough and establish a groundwork for the future advancement of gluten-free dietary options. PRACTICAL APPLICATION: The thorough analysis conducted in this experiment provides a theoretical framework for rice dough preparation during the fermentation process, addressing the dietary needs of individuals with coeliac disease and those following a gluten-free diet. This study also paves the way for the development of improved gluten-free rice products in future research pursuits., (© 2024 Institute of Food Technologists.)

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.)

Background: Grapholita molesta is an important and harmful fruit pest worldwide, with widespread feeding hosts. Trypsin, an indispensable hydrolytic digestive protease in the insect gut, is crucial in digestion, growth and development. We analyzed the characteristics of the trypsin-encoding genes, screened for the optimal dose of RNAi mediated by nanocarriers, and investigated various indices of larval growth and development of G. molesta., Results: Gut content (GC) and RNase A degraded double-stranded RNA (dsRNA), with a faster degradation rate at higher concentrations. Star polycation (SPc) nanomaterials protected dsGFP from degradation by anion-cation binding and did not migrate through agarose gel. The key conserved motifs of the trypsin-encoding genes were similar, exhibiting high homology with those in other lepidopteran insects. An interference efficiency of ≈70% was achieved with SPc nanomaterial-mediated RNA interference with 0.05 μg dsRNA. The efficiency of continuous interference was stable. Trypsin activity, body weight of 8-day-old larvae, pupal weight and emergence rate were significantly reduced, and the larval stage was significantly prolonged., Conclusion: The investigated trypsin gene is a key target gene in the growth and development of G. molesta. We investigated the efficiency and convenience of feeding SPc nanomaterials in a functional study of insects. Our results provide valuable data for the development of efficient trypsin-targeting pesticides. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Intradiscal injection is required to deliver therapeutic agents to the intervertebral disc (IVD) nucleus pulposus (NP). However, injectate leakage following needle retraction may result in decreased treatment efficacy and adverse side effects. While enzymatic digestion is a common research approach for simulating degeneration in healthy animal IVDs, contributions to the leakage phenomenon are unknown. In this study, bovine caudal discs were treated with injection into the NP of either a tris buffer control, collagenase (to primarily target collagen), or trypsin (to primarily target proteoglycans) and then injected with fluorescent saline using a through-puncture defect protocol. Pressure-volume records during injection were used to determine volume and pressure at leakage. Discs were then frozen, transected, and photographed to visualize injectate dispersion. Collagenase treatment resulted in a large increase in injectate dispersion, along with a decrease in injection pressure relative to control. Trypsin treatment resulted in a moderate increase in dispersion, with no associated effect on pressure. This study concludes that care should be taken when employing enzymatic digestion to simulate IVD degeneration, as NP tissue disruption may affect both retention and dispersion of subsequent therapeutic injections., (Copyright © 2024 by ASME.)

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.

The mechanism that triggers the progressive dysregulation of cell functions, inflammation, and breakdown of tissues during aging is currently unknown. We propose here a previously unknown mechanism due to tissue autodigestion by the digestive enzymes. After synthesis in the pancreas, these powerful enzymes are activated and transported inside the lumen of the small intestine to which they are compartmentalized by the mucin/epithelial barrier. We hypothesize that this barrier leaks active digestive enzymes (e.g. during meals) and leads to their accumulation in tissues outside the gastrointestinal tract. Using immune-histochemistry we provide evidence in young (4 months) and old (24 months) rats for significant accumulation of pancreatic trypsin, elastase, lipase, and amylase in peripheral organs, including liver, lung, heart, kidney, brain, and skin. The mucin layer density on the small intestine barrier is attenuated in the old and trypsin leaks across the tip region of intestinal villi with depleted mucin. The accumulation of digestive enzymes is accompanied in the same tissues of the old by damage to collagen, as detected with collagen fragment hybridizing peptides. We provide evidence that the hyperglycemia in the old is accompanied by proteolytic cleavage of the extracellular domain of the insulin receptor. Blockade of pancreatic trypsin in the old by a two-week oral treatment with a serine protease inhibitor (tranexamic acid) serves to significantly reduce trypsin accumulation in organs outside the intestine, collagen damage, as well as hyperglycemia and insulin receptor cleavage. These results support the hypothesis that the breakdown of tissues in aging is due to autodigestion and a side-effect of the fundamental requirement for digestion., Competing Interests: The authors own stock in Palisade Bio Inc. a company developing new treatments to protect gastrointestinal barrier function. No funding was obtained for this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 DeLano, Schmid-Schönbein. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.)

Aim: Molecular alterations of diabetic gastroenteropathy are poorly identified. This study investigates the effects of prolonged GABA supplementation on key protein expression levels of trypsin-1, PAR-1, PAR-2, PAR-3, PI3K, Akt, COX-2, GABAA, and GABAB receptors in the gastric tissue of type 2 diabetic rats (T2DM)., Method: To induce T2DM, a 3-month high-fat diet and 35 mg/kg of streptozotocin was used. Twenty-four male Wistar rats were divided into 4 groups: (1) control, (2) T2DM, (3) insulin-treated (2.5 U/kg), and (4) GABA-treated (1.5 g/kg GABA). Blood glucose was measured weekly. The protein expressions were assessed using western blotting. Histopathological changes were examined by H&E and Masson's staining., Results: Diabetic rats show reduced NOS1 and elevated COX-2 and trypsin-1 protein expression levels in gastric tissue. Insulin and GABA therapy restored the NOS1 and COX-2 levels to control values. Insulin treatment increased PI3K, Akt, and p-Akt and, decreased trypsin-1, PAR-1, PAR-2, and PAR-3 levels in the diabetic rats. Levels of GABAA and GABAB receptors normalized following insulin and GABA therapy. H&E staining indicated an increase in mucin secretion following GABA treatment., Conclusion: These results suggest that GABA by acting on GABA receptors may regulate the trypsin-1/PARs/Akt/COX-2 pathway and thereby improve complications of diabetic gastroenteropathy., (© 2024. Pleiades Publishing, Ltd.)

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.)

Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies., (© 2024 The Authors. ELECTROPHORESIS published by Wiley‐VCH GmbH.)

The serine protease chymotrypsin protects the pancreas against pancreatitis by degrading trypsinogen, the precursor to the digestive protease trypsin. Taking advantage of previously generated mouse models with either the Ctrb1 gene (encoding chymotrypsin B1) or the Ctrl gene (encoding chymotrypsin-like protease) disrupted, here we generated the novel Ctrb1-del × Ctrl-KO strain in the C57BL/6N genetic background, which harbors a naturally inactivated Ctrc gene (encoding chymotrypsin C). The newly created mice are devoid of chymotrypsin, yet the animals develop normally, breed well, and show no spontaneous phenotype, indicating that chymotrypsin is dispensable under laboratory conditions. When given cerulein, the Ctrb1-del × Ctrl-KO strain exhibited markedly increased intrapancreatic trypsin activation and more severe acute pancreatitis, relative to wild-type C57BL/6N mice. After the acute episode, Ctrb1-del × Ctrl-KO mice spontaneously progressed to chronic pancreatitis, whereas C57BL/6N mice recovered rapidly. The cerulein-induced pancreas pathology in Ctrb1-del × Ctrl-KO mice was highly similar to that previously observed in Ctrb1-del mice; however, trypsin activation was more robust and pancreatitis severity was increased. Taken together, the results confirm and extend prior observations demonstrating that chymotrypsin safeguards the pancreas against pancreatitis by limiting pathologic trypsin activity. In mice, the CTRB1 isoform, which constitutes about 90% of the total chymotrypsin content, is responsible primarily for the anti-trypsin defenses and protection against pancreatitis; however, the minor isoform CTRL also contributes to an appreciable extent. NEW & NOTEWORTHY Chymotrypsins defend the pancreas against the inflammatory disorder pancreatitis by degrading harmful trypsinogen. This study demonstrates that mice devoid of pancreatic chymotrypsins are phenotypically normal but become sensitized to secretagogue hyperstimulation and exhibit increased intrapancreatic trypsin activation, more severe acute pancreatitis, and rapid progression to chronic pancreatitis. The observations confirm and extend the essential role of chymotrypsins in pancreas health.

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.)

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.)

Kunitz-type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz-type trypsin inhibitors as identified by N-terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI-4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI-4 utilized a novel tryptophan-containing β1-β2 loop to bind trypsin. Site-direct mutagenesis confirmed the inhibitory role of this loop. DzTI-4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum-free conditions. DzTI-4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz-type trypsin inhibitors and provides clues for further application of these inhibitors., (© 2024 The Protein Society.)

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.)

Objective: The aim of this study was to investigate the effect of bovine trypsin on the adhesion and pH of dental plaque biofilms., Methods: A multispecies dental plaque biofilm model and a single-species dental plaque biofilm model were established in vitro. Three groups were tested: (1) blank control group (aseptic ultrapure water); (2) negative control group (1M Tris-HCl buffer, pH = 7.4); and (3) experimental group (bovine trypsin). Adhesion ability was measured using an automatic microplate reader and visualised by confocal laser scanning microscopy (CLSM). The pH was measured using a pH meter. The expression of gtfB, gtfC, and gtfD was analysed using quantitative real-time polymerase chain reaction., Results: Adhesion ability in the experimental group was significantly lower than that in the blank group and the negative control group (P < .05); readhesion ability in the experimental group was inhibited for a certain period of time (24-hour multispecies biofilms were inhibited from 4 to 8 hours, and the 48- and 72-hour multispecies biofilms were inhibited from 2 to 6 hours; P < .05). The decrease in pH was inhibited for a certain period of time (24-hour multispecies biofilms were inhibited from 2 to 8 hours, and the 48- and 72-hour multispecies biofilms were inhibited from 1 to 8 hours; P < .05). Expression levels of gtfB, gtfC, gtfD, and ldh in the experimental group were significantly lower than those in the blank group (P < .05)., Conclusions: Bacterial adhesion, and readhesion, decreasd pH, and expression of adhesion- and acid-related genes by Streptococcus mutans in biofilms could be reduced by bovine trypsin for a certain period of time., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

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).)

Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin concentration and exposure time., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)