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This work describes a novel automated and rapid method for bottom-up proteomics combining protein isolation with a micro-immobilised enzyme reactor (IMER). Crosslinking chemistry based on 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling was exploited to immobilise trypsin and antibodies onto customisable silica particles coated with carboxymethylated dextran (CMD). This novel silica-CMD solid-phase extraction material was characterised using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), conductometric titrations and enzymatic colorimetric assays. Micro-solid-phase extraction (μSPE) cartridges equipped with the modified CMD material were employed and integrated into an automated and repeatable workflow using a sample preparation workstation to achieve rapid and repeatable protein isolation and pre-concentration, followed by tryptic digestion producing peptide fragments that were identified by liquid chromatography mass spectrometry (LC-MS)., (© 2022. The Author(s).)

The N α-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BApNA) assay is widely used to quantify trypsin in mosquito midguts and is highly sensitive. BApNA is a chromogenic substrate for proteolytic enzymes such as trypsin and amidase. Hydrolysis of BApNA at the bond between the arginine and the p-nitroaniline moieties releases the chromophore p-nitroaniline, which is detected by colorimetric analysis. The intensity of the color is directly proportional to the amount of trypsin in the solution. Here, we present a trypsin measurement assay specifically using the BApNA substrate., (© 2023 Cold Spring Harbor Laboratory Press.)

Antihistamines and glucocorticoids are commonly used to treat allergy symptoms and the inflammatory conditions. In present study, the in-vitro binding interactions a glucocortikoid, dexamethasone/an antihistamine, pheniramine with TSN (TSN) secreted from pancreas to small intestine for protein digestion were investigated by fluorescence emission spectroscopy (FES), UV-Vis spectroscopy, synchronous fluorescence spectroscopy (SFS), CD spectroscopy, FT-IR and molecular modeling methods. Also, the effect of these drugs on the catalytic activity of trypsin (TSN) was determined. The fluorescence quenching experiments indicated that each drugs quenched the intrinsic fluorescence of TSN with their increased concentrations. The results of SFS and UV-Vis spectra proved the interaction of dexamethasone and pheniramine with TSN. CD spectra showed that the secondary structure of enzyme was altered in the presence of the drugs. All these spectroscopy results were validated and explained by molecular docking and molecular dynamic simulation (MD) studies. The IC 50 values were determined as 0.0049 mM and 0.0038 mM for dexamethasone and pheniramine, respectively. So, both drugs have inhibition effect on the catalytic activity of TSN. The results of this study can provide valuable information in the field of pharmacokinetics and pharmacodynamics.Communicated by Ramaswamy H. Sarma.

Currently, the bottom-up approach, in which proteins are digested by enzymes such as trypsin prior to mass spectrometry, is the mainstream approach in mass spectrometer-based proteomics. In this approach, the enzymatic digestion process strongly affects the reproducibility of protein identification and quantification. Here, we quantitatively evaluated the enzymatic digestion of proteins under various conditions by quantitative proteomics using data-independent acquisition and found that proteins precipitated with acetone after solubilization with SDS were fully digestible without re-solubilization. This result implies that organic solvent treatment makes cells amenable to trypsin digestion. Direct trypsin digestion of methanol-fixed cells achieved the same digestion efficiency and quantitative reproducibility as the conventional method. Furthermore, this method was found to be equally applicable to mouse liver samples. The establishment of this method indicates that the sample preparation process in bottom-up proteomics can be simplified while maintaining high digestion efficiency and is expected to become a general method for sample preparation in bottom-up proteomics in the future., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The subtilisin-like macrocyclase PatGmac is produced by the marine cyanobacterium Prochloron didemni . This enzyme is involved in the last step of the biosynthesis of patellamides, a cyanobactin type of ribosomally expressed and post-translationally modified cyclic peptides. PatGmac recognizes, cleaves, and cyclizes precursor peptides after a specific recognition motif comprised of a C-terminal tail with the sequence motif -AYDG. The result is the native macrocyclic patellamide, which has eight amino acid residues. Macrocyclase activity can be exploited by incorporating that motif in other short linear peptide precursors, which then are formed into head-to-tail cyclized peptides. Here, we explore the possibility of using PatGmac in the cyclization of peptides larger than the patellamides, namely, the PawS-derived peptide sunflower trypsin inhibitor-1 (SFTI-1) and the cyclotide kalata B1. These peptides fall under two distinct families of disulfide constrained macrocyclic plant peptides. They are both implicated as scaffolds for drug design due to their structures and unusual stability. We show that PatGmac can be used to efficiently cyclize the 14 amino acid residue long SFTI-1, but less so the 29 amino acid residue long kalata B1.

In the current study, the interaction of minocycline hydrochloride (MC) and trypsin (TRP) was studied using fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, UV-Vis spectroscopy, and molecular docking simulation techniques. The results show that the fluorescence quenching of trypsin at different degrees can be caused by minocycline hydrochloride at different temperatures. According to the Stern-Volmer equation, the fluorescence quenching type was static quenching. By calculating critical distance, we concluded that there is a possibility of non-radiative energy transfer between minocycline hydrochloride and trypsin. The effect of minocycline hydrochloride on the secondary structure of trypsin was demonstrated using ultraviolet spectroscopy. Synchronous fluorescence spectroscopy showed that minocycline hydrochloride could bind to tryptophan residues in trypsin, resulting in corresponding changes in the secondary structure of trypsin. Three-dimensional fluorescence spectroscopy showed that minocycline hydrochloride had a particular effect on the microenvironment of trypsin that led to changes in the secondary structure of trypsin. The molecular docking technique demonstrated that the binding of minocycline hydrochloride and trypsin was stable. Circular dichroism showed that the secondary structure of trypsin could be changed by minocycline hydrochloride.

Two series of macrocyclic plasmin inhibitors with a C-terminal benzylamine group were synthesized. The substitution of the N-terminal phenylsulfonyl group of a previously described inhibitor provided two analogues with sub-nanomolar inhibition constants. Both compounds possess a high selectivity against all other tested trypsin-like serine proteases. Furthermore, a new approach was used to selectively introduce asymmetric linker segments. Two of these compounds inhibit plasmin with K i values close to 2 nM. For the first time, four crystal structures of these macrocyclic inhibitors could be determined in complex with a Ser195Ala microplasmin mutant. The macrocyclic core segment of the inhibitors binds to the open active site of plasmin without any steric hindrance. This binding mode is incompatible with other trypsin-like serine proteases containing a sterically demanding 99-hairpin loop. The crystal structures obtained experimentally explain the excellent selectivity of this inhibitor type as previously hypothesized., (© 2023 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The combination of acoustically levitated droplets, mid-IR laser evaporation, and subsequent post-ionization by secondary electrospray ionization was applied for monitoring the enzymatic digestion of various proteins. Acoustically levitated droplets are an ideal, wall-free model reactor, readily allowing compartmentalized microfluidic trypsin digestions. Time-resolved interrogation of the droplets yielded real-time information on the progress of the reaction and thus provided insights into reaction kinetics. After 30 min of digestion in the acoustic levitator, the obtained protein sequence coverages were identical to the reference overnight digestions. Importantly, our results clearly demonstrate that the applied experimental setup can be used for the real-time investigation of chemical reactions. Furthermore, the described methodology only uses a fraction of the typically applied amounts of solvent, analyte, and trypsin. Thus, the results exemplify the use of acoustic levitation as a green analytical chemistry alternative to the currently used batch reactions.

The detection of trypsin is significantly important for both clinical diagnosis and disease treatment. In this study, an innovative multicolor sensor for trypsin detection has been established based on the regulation of the peroxidase activity of bovine serum albumin-coated gold nanoclusters (BSA-Au NCs) and efficient etching of gold nanobipyramids (Au NBPs). BSA-Au NCs have slight peroxidase enzyme activity and can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate TMB + , while trypsin can hydrolyze BSA ligands on the surface of BSA-Au NCs, thus exposing more catalytic active sites of BSA-Au NCs and resulting in the enhancement of the peroxidase activity of BSA-Au NCs, hence more TMB + is generated. Under acidic conditions, TMB + can etch Au NBPs efficiently, consequently affecting the aspect ratio of Au NBPs accompanied by the ultraviolet-visible (UV-vis) spectra blue shifting of the system. Furthermore, this also results in color variations that can be distinguished and recognized by naked eyes without any expensive and sophisticated instruments. This multicolor sensor has an available linear relationship with the logarithm of the trypsin concentration in the range of 0.1-100 μg/mL, and the detection limit is 0.045 μg/mL. The designed sensor has been used to detect the concentration of trypsin in human serum samples from healthy individuals and pancreatitis patients with satisfactory results.

Kazal inhibitors hold high potential as scaffolds for therapeutic molecules, taking advantage of the easily exchangeable canonical binding loop. Different Kazal inhibitor backbones have been suggested to be therapeutically useful, but the impact of different Kazal-like scaffolds on binding properties is still largely unknown. Here, we identified trypsin-targeting human serine protease inhibitor Kazal type 1 (SPINK1) homologues in different mammalian species that cluster in two P2-P1 combinations, implying the coevolution of these residues. We generated loop exchange variants of human SPINK1 for comparison with Kazal inhibitors from related species. Using comprehensive biophysical characterization of the inhibitor-enzyme interactions, we found not only affinity but also pH resistance to be highly backbone-dependent. Differences are mostly observed in complex stability, which varies by over one order of magnitude. We provide clear evidence for high backbone dependency within the Kazal family. Hence, when designing Kazal inhibitor-based therapeutic molecules, testing different backbones after optimizing the canonical binding loop can be beneficial and may result in increased affinity, complex stability, specificity, and pH resistance.

As a kind of novel functional material, graphene-related nanomaterials (GRMs) have great potentials in industrial and biomedical applications. Meanwhile, the production and wide application of GRMs will increase the risk of unintended or intentional oral exposure to human beings, attracting safety concerns about their biological fates and toxicological effects. The normal enzymatic activity of digestive enzymes is essential for the proper functioning of the gastrointestinal tract system. However, whether and how orally entered GRMs and their surface groups affect digestive enzymes' activity are still scarce. In this paper, we systematically studied the effects of graphene oxide (GO), graphene modified with hydroxyl groups (OH-G), carboxyl groups (COOH-G), and amino groups (NH 2 -G) on enzymatic activity of three typical digestive enzymes (pepsin, trypsin, and α-pancreatic amylase). The results showed that the activity of trypsin and α-pancreatic amylase could be greatly changed after GRMs incubation in a surface chemistry dependent manner, while the activity of pepsin was not affected. To elucidate the mechanisms at the molecular level, the interactions between trypsin and GRMs were studied by spectrometry, thermophoresis, and computational simulation approaches, and the key roles of surface chemistry of GRMs in tailoring the activity of trypsin were finally figured out. GO allosterically inhibited trypsin's activity in the non-competitive manner because of the conformation transition induced by the intensive interactions. COOH-G could effectively hamper enzymatic activity of trypsin in the competitive manner by blocking the active catalytic pocket. As for NH 2 -G and OH-G, they had little impact on the activity of trypsin due to the weak binding affinity or limited conformational change. Our findings not only indicate surface chemistry plays an important role in tailoring the effects of GRMs on the activity of digestive enzymes but also provide new insights for understanding the oral safety of nanomaterials from daily products and the environment., 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 © 2022 Elsevier Inc. All rights reserved.)

In this paper, Ti 3 C 2 quantum dots (Ti 3 C 2 QDs) were synthesized by simply treating Ti 3 C 2 MXene powder with acid and base via hydrothermal method. Ti 3 C 2 QDs exhibited superior fluorescence property and were used for the fluorescent imaging of living HeLa cells successfully. In order to evaluate the influence of Ti 3 C 2 QDs on protease with specific biological functions, binding interaction of Ti 3 C 2 QDs with trypsin was studied comprehensively and deeply through spectroscopic strategies and molecular modeling technique. The intrinsic fluorescence of trypsin was spontaneously quenched by Ti 3 C 2 QDs through static quenching mode under van der Waals interaction force, and Ti 3 C 2 QDs bound with the inactive residue domain of trypsin firmly with stoichiometric ratio of 1:1. Ti 3 C 2 QDs induced the microenvironmental variation of the amino acid residues in trypsin, reducing the thermal stability of trypsin significantly. Gel electrophoresis experiments and microscopic imaging experiments demonstrated that Ti 3 C 2 QDs inhibited the enzymatic activity of trypsin on the digestion of human serum albumin and HeLa cells obviously. These results revealed not only the deep interaction mechanism between Ti 3 C 2 QDs and protease but also the influence of Ti 3 C 2 QDs on the enzymatic activity of trypsin, paving the way for the safe biological application of Ti 3 C 2 QDs in the diagnosis and the therapy of protease-related diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The interactions between metabolites and proteins constitute crucial events in cell signaling and metabolism. In recent years, large-scale proteomics techniques have emerged to identify and characterize protein-metabolite interactions. However, their implementation in plants is generally lagging behind, preventing a complete understanding of the regulatory mechanisms governing plant physiology. Recently, a novel approach to identify metabolite-binding proteins, namely, limited proteolysis-coupled mass spectrometry (LiP-MS), was developed originally for microbial proteomes. Here, we present an adapted and accessible version of the LiP-MS protocol for use in plants. Plant proteomes are extracted and incubated with the metabolite of interest or control treatment, followed by a limited digestion by a nonspecific/promiscuous protease. Subsequently, a conventional shotgun proteomics sample preparation is performed including a complete digestion with the sequence-specific protease trypsin. Finally, label-free proteomics analysis is applied to identify structure-dependent proteolytic patterns corresponding to protein targets of the specific metabolite and their binding sites. Given its amenability to relatively high throughput, the LiP-MS approach may open a potent avenue for the discovery of novel regulatory mechanisms in plant species., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cassia siamea is a nonedible legume belonging to Fabaceae. The seed of C. siamea contains ~16% of protein. The study reports the biochemical characterization of purified novel serine protease inhibitor from seeds of C. siamea, aimed with assessing the anti-inflammatory activity. The seed extract was subjected to ammonium sulfate precipitation followed by fast protein liquid chromatography (FPLC)-anion exchange chromatography and affinity-chromatography to obtain a relative pure protease inhibitor. Thirty-fivefold purification with the specific activity of 250 U/mg of trypsin inhibitory unit was obtained. The characterization of protease inhibitor for optimum temperature, pH, and metal ions were measured using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) assay and casein zymogram. The C. siamea trypsin inhibitor (CsTI) has a relative molecular mass of 25.540 kDa. Purified CsTI and Dolichos biflorus were tested for anti-inflammatory efficacy against A549 and RAW264.7 cell lines. The inhibitory activity of both purified inhibitors are comparable and are potent toward anti-inflammatory activity. The purified inhibitor shows to be a promising candidate as anti-inflammatory agent by targeting the serine proteases., (© 2022 John Wiley & Sons Ltd.)

Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification., Competing Interests: Conflict of interest The authors declare no competing interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

To regulate nanostructure synthesis is of crucial importance for developing various applications, including catalysis, bioanalysis, and optical devices. Herein, the morphology and peroxidase (POD)-mimicking activity of peptide-templated copper nanoassemblies (Cu NAs) are regulable with peptide types. The Cu NAs templated with peptide containing single cysteine are uniform nanoclusters with strong POD-like activity. However, the Cu NAs templated with peptide containing two cysteines are fusiform-like with very weak POD-like activity. Unexpectedly, the POD-like activity of Cu NAs templated with peptide containing two cysteines with lysine between the cysteines is significantly enhanced when trypsin is incubated, which is unchanged for the Cu NAs templated with peptide containing two cysteines without lysine between the cysteines. The remarkably enhanced POD-mimicking activity originates from trypsin specifically shearing the peptide bond on the lysine, thereby allowing the aggregated Cu NAs to unravel into individual nanoclusters. Therefore, a robust colorimetric sensing platform was constructed for sensitive and selective detection of trypsin, which showed a linear concentration range of 3-1000 nM and a detection limit of 0.82 nM (S/N = 3). More interestingly, featured by trypsin inhibitor restraining trypsin activity, it enabled us to screen trypsin inhibitors as well. Subsequently, the developed assay was applied to detect trypsin in serum samples with good accuracy and reproducibility. Thus, this strategy shows great potential application in the clinic for diagnosis of trypsin-indicating diseases as well as the screening of trypsin inhibitor-based anti-cancer drugs.

In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.

For the understanding of pathological states of bone tissues in oral surgery, it would be desirable to have the possibility to simulate these processes on bone cell models in vitro. These cultures, similarly to bone tissues, contain numerous proteins entrapped in the insoluble matrix. The major goal of this study was to verify whether a method based on direct in-matrix protein digestion could be suitable for the discrimination between different induced pathological states of bone cell models cultivated in vitro. Using in-sample specific protein digestion with trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released peptides, 446 proteins (in average per sample) were identified in a bone cell in vitro model with induced cancer, 440 proteins were found in a model with induced inflammation, 451 proteins were detected in control in vitro culture, and 491 proteins were distinguished in samples of vestibular laminas of maxillary bone tissues originating from six different patients. Subsequent partial least squares - discrimination analysis of obtained liquid chromatography-tandem mass spectrometry data was able to discriminate among in vitro cultures with induced cancer, with induced inflammation, and control cultivation. Thus, the direct in-sample protein digestion by trypsin followed by liquid chromatography-tandem mass spectrometry analysis of released specific peptide fragments from the insoluble matrix and mathematical analysis of the mass spectrometry data seems to be a promising tool for the routine proteomic characterization of in vitro human bone models with induced different pathological states., (© 2022 Wiley-VCH GmbH.)

Acid Yellow 17 is a kind of azo dye used in food, textile, and cosmetics. Several studies explain the toxicity of azo dye for our body, but one could not find further information about the effects of these dyes on human macromolecules. In the current study, the interaction of AY17 with trypsin is investigated using several techniques. The UV analysis displayed that the absorption of trypsin could be decreased in the presence of this color. The fluorescence investigation indicated that a static form of quenching happens, and a 50% decrease in the fluorescence intensity, also showed the Vander Waals and hydrogen bond are the main forces in the interaction of this color and trypsin. Furthermore, we can observe that the T m point of trypsin decreases from 46.5 to 42. On the other hand, the CD results were indicated that the interaction of this color with trypsin could decrease the percent of turn, coil and α-helix in trypsin structure. The computational study was undertaken to obtain more information about the interaction between trypsin and AY17. The results were in agreement with the experimental investigation and indicated that the interaction between this color and trypsin leads to less compactness in the trypsin structure., (Copyright © 2022. Published by Elsevier B.V.)

Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimer's. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.

Purpose: Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins., Methods: The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP., Results: Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates., Conclusions: The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A new cationic indolium based styryl dye (Ci) as a fluorescent probe was synthesized and its anions selectivity/sensitivity properties/molecular interactions with protease enzymes (pepsin/trypsin) and ctDNA has been studied by spectroscopic and computational methods. The fluorescence measurements at different temperatures indicated that quenching mechanism of enzymes by Ci was static. ΔH and ΔS data pointed out electrostatic/hydrophobic interactions with pepsin, and also hydrogen bonds/van der Waals forces with trypsin of Ci. According to Förster's non-radiative energy transfer, binding distances (r) were calculated as 3.53/3.27 nm for pepsin/trypsin. It was also investigated that groove binding is effective in interaction with ctDNA. The results were supported with molecular docking analyzes which have same tendency. Ci has been demonstrated hypsochromic effect with a decrease in polarity of solvents and it showed highly selective colorimetric and fluorometric sensing behavior for cyanide in organic solvent and in aqueous solution. 1 H NMR titration was performed to examine the interaction mechanism between Ci and cyanide. The LOD values of cyanide ion were reported as 4.87 × 10 -9  M and 9.70 × 10 -7  M in DMSO and DMSO/H 2 O binary mixture, respectively. In addition, sensitivity of Ci as a chemosensor to cyanide was investigated in bitter almond samples., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin-activated form involves a major conformational change of the N-terminal Domain I, which is remodelled into a tetrameric coiled-coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C-t Domains IV and V to sustain the conformational change necessary for toxicity., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

In this work, a microwave assisted method was developed for synthesis of red fluorescent copper nanoclusters (NCs) using trypsin as a template (trypsin-Cu). The as-synthesized trypsin-Cu NCs are stable and water soluble, exhibiting fluorescence emission at 657 nm when excited at 490 nm. The as-prepared red-emitting trypsin-Cu NCs were characterized by using several analytical techniques such as ultraviolet-visible (UV-Vis) and fluorescence, fluorescence lifetime, Fourier transform infrared, and X-ray photoelectron spectroscopic techniques. Red-emitting trypsin-Cu NCs acted as a nanosensor for sensing both Pb 2+ and Hg 2+ ions through fluorescence quenching. Using this approach, good linearities are observed in the range of 0.1-25 and of 0.001-1 μM with the lower limit of detection of 14.63 and 56.81 nM for Pb 2+ and Hg 2+ ions, respectively. Trypsin-Cu NCs-based fluorescence assay was successfully applied to detect both Hg 2+ and Pb 2+ ions in water and tobacco samples.

The process of ligand-protein unbinding is crucial in biophysics. Water is an essential part of any biological system and yet, many aspects of its role remain elusive. Here, we simulate with state-of-the-art enhanced sampling techniques the binding of Benzamidine to Trypsin which is a much studied and paradigmatic ligand-protein system. We use machine learning methods to determine efficient collective coordinates for the complex non-local network of water. These coordinates are used to perform On-the-fly Probability Enhanced Sampling simulations, which we adapt to calculate also the ligand residence time. Our results, both static and dynamic, are in good agreement with experiments. We find that the presence of a water molecule located at the bottom of the binding pocket allows via a network of hydrogen bonds the ligand to be released into the solution. On a finer scale, even when unbinding is allowed, another water molecule further modulates the exit time., (© 2022. The Author(s).)

Specific salts effect is well-known on stability and solubility of proteins, however, relatively limited knowledge is known regarding the effect on catalytic properties of enzymes. Here, we examined the effect of four sodium anions on thermal stability and catalytic properties of trypsin and binding of the fluorescent probe, p-aminobenzamidine (PAB), to the enzyme. We show that the specific anions effect on trypsin properties agrees with the localization of the anions in the Hofmeister series. Thermal stability of trypsin, T m , the affinity of the fluorescent probe to the binding site, K d , and the rate constant, k cat , of trypsin-catalyzed hydrolysis of the substrate N-benzoyl-L-arginine ethyl ester (BAEE) increase with increasing kosmotropic character of anions in the order: perchlorateM , decreases. Correlations between the values of T m , K d for PAB, k cat , and K M for BAEE in the presence of 1 M studied salts suggest interrelation among these parameters of the enzyme. Global stabilization as well as increased rigidity of trypsin is accompanied by strengthening of interaction with fluorescent probe PAB and in accordance with decreasing values of K M for the substrate BAEE. Strong correlations between parameters characterizing the trypsin properties with the charge densities of anions clearly indicate direct electrostatic interaction as a basis of the specific anion effect on the conformational and functional properties of the enzyme., (Copyright © 2022. Published by Elsevier B.V.)

Trypsin, the most abundant pancreatic protein, aids in protein digestion by hydrolysis and exhibits aggregation propensity in presence of alcohol, which can further lead to pancreatitis and eventually pancreatic cancer. Herein, by several experimental and theoretical approaches, we unearth the inhibition of alcohol-induced aggregation of Trypsin by macrocyclic cavitand, β-cyclodextrin (β-CD). β-CD interacts with the native protein and shows inhibitory effect in a dose dependent manner. Moreover, the secondary structures and morphologies of Trypsin in presence of β-CD also clearly emphasize the inhibition of fibril formation. From Fluorescence Correlation Spectroscopy, we observed an enhancement in diffusion time of Nile Red with ∼2.5 times increase in hydrodynamic radius, substantiating the presence of fibrillar structure. Trypsin also shows reduction in its functional activity due to alcohol-induced aggregation. Our simulation data reports the probable residues responsible for fibril formation, which was validated by molecular docking studies., (© 2022 Wiley-VCH GmbH.)

Accurate genome annotation, the foundation of life science research in the genome era, is hampered by limited known gene models, nonstandard start codons, and the limited homology of annotated genes in other organisms. LysargiNase mirrors trypsin at the cleavage sites, providing the opportunity to identify peptides other than tryptic peptides. In this study, we used an in-house developed acetylated LysargiNase (Ac-LysargiNase) with higher activity and stability in non-pathogenic Mycolicibacterium smegmatis MC 2 155 to supplement the widely used trypsin in proteomic studies. We identified 27,582 peptides from 3844 annotated proteins and 332 novel genome search-specific peptides (GSSPs). Among these GSSPs, 88 peptides were annotated in another M.smegmatis genome database, and 41 were verified as novel peptides by predicted theoretical spectra and their corresponding 15 N-labeling spectra. Further analysis revealed that 17 verified GSSPs corrected the N-terminus of the 13 annotated genes. The other 24 verified GSSPs helped identify 17 novel open reading frames (ORFs) missed in previously annotated M. smegmatis genomes. Among these novel ORFs, four relatively small proteins with amino acid residues less than 100 and three were precisely identified with C-terminal peptides. Ac-LysargiNase helps with genome reannotation by identifying new genes and events in proteogenomic studies. SIGNIFICANCE: Correct genomic annotation is vital in the field of life sciences. The nonstandard start codons seriously affect the confirmation of the translation initiation sites (TISs) of an open reading frame (ORF), and unknown structural genes are easily missed in automated gene prediction. Although proteogenomics presents new avenues for validating gene expression and gene structure refinement based on conventional tryptic peptides, determining the TISs and potential encoding genes is complicated. Thus, validation of TISs and encoding ORFs is crucial and urgent. Therefore, we recommend Ac-LysargiNase, a mirror enzyme of trypsin that can identify additional novel peptides for N-terminal correction and ORF identification., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 μs. Further investigation of peptides resulting from microdroplet digestion (MD) would be necessary to evaluate it as an alternative to the conventional bulk digestion for bottom-up and biotherapeutic protein characterization. Herein we examined and compared protein tryptic digestion in both MD and bulk solution. In the case of MD of β-lactoglobulin B, the preservation of long peptides was observed due to the short digestion time. In addition, MD is applicable to digest both high- and low-abundance proteins in mixture. In the case of digesting NIST 8671 mAb antibody containing a low level of commonly encountered host cell protein (HCP) PLBL2 (mAb:PLBL2 = 100:1 by weight), MD produced lower levels of digestion-induced chemical modifications of asparagine/glutamine deamidation, compared with overnight digestion. No significant difference between MD and bulk digestion was observed in terms of trypsin digestion specificity based on examination of semi- and unspecific-cleaved peptides. Our study suggests that MD, a fast digestion approach, could be adopted for bottom-up proteomics research and for peptide mapping of mAbs to characterize site-specific deamidation and glycosylation, for the purpose of development of biopharmaceuticals.

For the past few years, multidimensional liquid chromatography-mass spectrometry (LC-MS) systems have been commonly used to characterize post-translational modifications (PTMs) of therapeutic antibodies (mAbs). In most cases, this is performed by fractionation of charge variants by ion-exchange chromatography and subsequent online LC-MS peptide mapping analysis. In this study, we developed a multidimensional ultra-performance-liquid-chromatography-mass spectrometry system (mD-UPLC-MS/MS) for PTM characterization and quantification, allowing both rapid analysis and decreased risk of artificial modifications during sample preparation. We implemented UPLC columns for peptide mapping analysis, facilitating the linkage between mD-LC and routine LC-MS workflows. Furthermore, the introduced system incorporates a novel in-parallel trypsin and LysC on-column digestion setup, followed by a combined peptide mapping analysis. This parallel digestion with different enzymes enhances characterization by generating two distinct peptides. Using this approach, a low retentive ethylene oxide adduct of a bispecific antibody was successfully characterized within this study. In summary, our approach allows versatile and rapid analysis of PTMs, enabling efficient characterization of therapeutic molecules.

The interaction of one anticancer drug (caffeic acid phenethyl ester; CAPE) with three proteases (trypsin, pepsin and α-chymotrypsin) has been investigated with multispectral methods and molecular docking. As an active components in propolis, the findings are of great benefit to metabolism, design, and structural modification of drugs. The results show that CAPE has an obvious ability to quench the trypsin, pepsin, or α-chymotrypsin fluorescence mainly through a static quenching procedure. Trypsin has the largest binding affinity to CAPE, and α-chymotrypsin has the smallest binding affinity to CAPE. The data obtained from thermodynamic parameters and molecular docking prove that the spontaneously interaction between CAPE and each protease is mainly due to a combination of van der Waals (vdW) force and hydrogen bond (H-bond), controlled by an enthalpy-driven process. The binding force, strength, position, and the number of H-bond are further obtained from the results of molecular docking. Through ultraviolet spectroscopy, dynamic light scattering and circular dichroism experiments, the change in the protease secondary structure induced by CAPE was observed. Additionally, the addition of protease had a positive effect on the antioxidative activity of CAPE, and α-chymotrypsin has the greatest effect on the removal of 2,2-diphenyl-1-picrylhydrazyl free radicals by CAPE., (© 2022 John Wiley & Sons Ltd.)

In this study, the interaction between gold nanoparticles (AuNPs) and proteins (including lysozyme, trypsin, pepsin, γ-globulin and hemoglobin) was investigated by UV-visible absorption spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and protein activity assay. AuNPs was synthesized using reduction of HAuCl 4 with sodium citrate. The formation of AuNPs was confirmed from the characteristic surface plasmon resonance band at 521 nm and transmission electron microscopy revealed the average particle size was about 10 nm. The results reveal that AuNPs can interact with proteins to form a "protein corona (PC)", but the protein concentration required to form a relatively stable PC is not the same. The quenching mechanism of proteins by AuNPs is arisen from static quenching. The binding constants of AuNPs with proteins are in the range from 10 6 to 10 10  L mol -1 , and the order is pepsin > γ-globulin > hemoglobin > trypsin > lysozyme at 298 K. Van der Waals forces and hydrogen bonds are the main forces for the lysozyme-AuNPs system. The interaction between trypsin/pepsin/γ-globulin/hemoglobin and AuNPs is mainly by hydrophobic interaction. The addition of AuNPs has an effect on the secondary structure of proteins as confirmed from CD spectra. The change in secondary structure of different proteins is different and seems to have little relation with the binding constant. The activity of lysozyme/trypsin/pepsin decreases with the addition of AuNPs., 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 © 2022 Elsevier B.V. All rights reserved.)

The interaction between myricetin and dihydromyricetin with trypsin, α-chymotrypsin and lysozyme was investigated using multispectral and molecular docking methods. The results of fluorescence quenching revealed that myricetin and dihydromyricetin could quench the intrinsic fluorescence of three different proteinases through a static quenching procedure. The binding constant and number of binding sites at different temperatures were measured. The thermodynamic parameters obtained at different temperatures showed van der Waals interactions and hydrogen bonds played the main roles in the interaction of myricetin with trypsin and lysozyme, hydrophobic force was dominant both in myricetin with α-chymotrypsin interaction and dihydromyricetin with trypsin and lysozyme interaction, as for the electrostatic forces, it was mainly the driving force in dihydromyricetin binding to α-chymotrypsin. There was non-radiative energy transfer between three proteinases and myricetin or dihydromyricetin with high probability. The microenvironment of trypsin, α-chymotrypsin and lysozyme is changed. The docking studies revealed that myricetin and dihydromyricetin entered the hydrophobic cavity of three proteinases and formed hydrogen bonds. The binding affinity of myricetin or dihydromyricetin is different with the trypsin, α-chymotrypsin and lysozyme due to the different molecular structure., (© 2022 John Wiley & Sons Ltd.)

A general strategy for improving the zymolytic stability against proteases is reported. Carboxylatopillar[6]arene (CP6A) could effectively bind arginine and arginine-containing peptides, thereby improving the stability of angiotensin peptides in the presence of trypsin by the complexation of the side chain of the arginine residue.

Examination of a series of naturally-occurring trypsin inhibitor proteins, led to identification of a set of three residues (which we call the "interface triplet") to be determinant of trypsin binding affinity, hence excellent templates for small molecule mimicry. Consequently, we attempted to use the Exploring Key Orientation (EKO) strategy developed in our lab to evaluate small molecules that mimic the interface triplet regions of natural trypsin inhibitors, and hence potentially might bind and inhibit the catalytic activity of trypsin. A bis-triazole scaffold ("TT-mer") was the most promising of the molecules evaluated in silico . Twelve such compounds were synthesized and assayed against trypsin, among which the best showed a K d of 2.1 μM. X-ray crystallography revealed a high degree of matching between an illustrative TT-mer's actual binding mode and that of the mimics that overlaid the interface triplet in the crystal structure. Deviation of the third side chain from the PPI structure seems to be due to alleviation of an unfavorable dipole-dipole interaction in the small molecule's actual bound conformation.

Protein complexes are involved in a variety of biological activities. Accurate and comprehensive characterization of the structures and interactions of protein complexes is crucial in determining their biological functions. Chemical cross-linking coupled with mass spectrometry (CXMS) is an emerging investigative technique for protein complexes. CXMS enables the sensitive high-throughput analysis of protein complexes without the requirements of molecular weight and purification. These attributes have spurred the increased use of CXMS for the structure and interaction characterization of purified protein complexes and complicated cell lysate samples. CXMS utilizes chemical cross-linking reagents to covalently connect two reactive amino acids in or between proteins that are spatially close to each other. Subsequently, the cross-linked proteins are digested into cross-linked peptides, followed by LC-MS/MS analysis, as well as database searching to provide cross-linking information for the composition, interaction, and structural site distance restrictions of protein complex identification. Therefore, identification of cross-linked sites has a decisive influence on the characterization of protein complexes. This identification is limited by the unsatisfactory quality of the cross-linked peptide spectrum. Insufficient b/y fragment ions and poor continuity of amino acid sequence matching lead to low coverage and accuracy of cross-linked site identification. Based on the complementary feature of mirror-cutting digestion, an orthogonal digestion strategy based on LysargiNase combined with trypsin was developed in this study. Trypsin is the most commonly utilized digestion enzyme in proteomics, with extremely high enzyme activity and specificity. Trypsin generates C-terminally charged peptides after lysine (K) and arginine (R). LysargiNase is a mirror protease complementary to trypsin that cleaves before the K and R residues. This generates peptides with an N-terminal positively-charged residue. Owing to the different physical and chemical micro-environments of the cross-linked peptides digested by LysargiNase and trypsin, the behavior of their detection ability in MS analysis is diverse. Using the orthogonal digestion strategy, both simple and complicated cross-linked samples were analyzed in this study. For the analysis of bovine serum albumin (BSA), 291 pairs of non-redundant cross-linked sites were obtained, of which 216 pairs of cross-linked sites were provided by trypsin digestion, whereas 75 pairs of cross-linked sites were exclusively supplied by LysargiNase digestion. Except for the 35% increase in the number of identified cross-linked sites, 32% of the spectra of the commonly identified cross-linked peptides have better quality with more b-type fragment ions and consecutive sequence matching. Furthermore, for the Escherichia coli sample, 726 pairs of cross-linked sites were obtained in total, among which, 624 and 274 pairs were identified from trypsin and LysargiNase digestion, respectively. LysargiNase digestion yielded 120 individual cross-linked sites, which resulted in a 16% increase in single trypsin digestion. Consistent with the BSA sample, the quality was improved in 35% of the spectra of commonly identified cross-linked peptides. Corresponding to the identified cross-linked peptides, 242 structural constraints with 607 pairs of intra-cross-linked sites and 29 sets of protein-protein interactions with 119 pairs of inter-cross-linked sites were obtained. The collective results demonstrated that, mirror cutting-assisted orthogonal digestion strategy could significantly increase the number of identified fragment ions and amino acid sequences matching the continuity of the spectra by contributing b-and y-type ions, respectively. This improved the accuracy and coverage of cross-linked peptide identification. The findings additionally demonstrate the superiority of our method in the accurate identification of the cross-linked peptide spectra and the increased number of identified cross-linked sites. In a word, this method is expected to provide new insights for the large-scale and highly accurate characterization of protein complexes.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

A 65-day growth trial was conducted to investigate the dietary protein requirements for Culter mongolicus fingerlings. Isolipidic and isoenergetic diets were formulated with five dietary protein levels (32%, 37%, 42%, 47%, and 52%). Each diet was assigned to triplicate groups of 70 C. mongolicus fingerlings (0.99±0.08 g). The results indicated that weight gain and specific growth rate (SGR) increased with increasing dietary protein levels up to 47%. The activities of intestinal trypsin and lipase were the lowest in the 32% protein and 52% protein groups, while amylase activity reduced markedly in the 47% protein group. These results suggest that different dietary protein levels may cause different transformations of nutrients. The activities of superoxide dismutase (SOD) and lysozyme were not affected by varying dietary protein levels, except for those in the 32% protein group. In contrast, the content of malondialdehyde (MDA) increased with increasing dietary protein levels and reaching a maximum in the 52% protein group, suggesting that MDA accumulation depends on the protein concentration and the potential oxidative stress. Taken together, based on the broken-line analysis of SGR, we recommended the optimum dietary protein for C. mongolicus fingerlings to be 48.97%~49.31%., Competing Interests: The authors have declared that no competing interests exist.

For decades, structural analysis of proteins have received considerable attention, from their sequencing to the determination of their 3D structures either in the free state (e.g., no host-guest system, apoproteins) or (non)covalently bound complexes. The elucidation of the 3D structures and the mapping of intra- and intermolecular interactions are valuable sources of information to understand the physicochemical properties of such systems. X-ray crystallography and nuclear magnetic resonance are methods of choice for obtaining structures at the atomic level. Nonetheless, they still present drawbacks which limit their use to highly purified systems in a relatively high amount. On the contrary, mass spectrometry (MS) has become a powerful tool thanks to its selectivity, sensitivity, and the development of structural methods both at the global shape and the residue level. The combination of several MS-based methods is mandatory to fully assign a putative structure in combination with computational chemistry and bioinformatics. In that context, we propose a strategy which complements the existing methods of structural studies (e.g., circular dichroism, hydrogen/deuterium exchange and cross-links experiments, nuclear magnetic resonance). The workflow is based on the collection of structural information on proteins from the apparition rates and the time of appearance of released peptides generated by a protease in controlled experimental conditions with online detection by electrospray high-resolution mass spectrometry. Nondenaturing, partially or fully denatured proteins were digested by the enzymatic reactor, i.e., β-lactoglobulin, cytochrome c , and β-casein. The collected data are interpreted with regard to the kinetic schemes with time-dependent rates of the enzymatic digestion established beforehand, considering kinetics parameters in the Michaelis-Menten formalism including k cat (the turnover number), k 1 (formation of the enzyme-substrate complex), k -1 (dissociation of the enzyme-substrate complex), k off (local refolding of the protein around the cleavage site), and k on (local unfolding of the protein around the cleavage site). Solvent-accessible surface analysis through digestion kinetics was also investigated. The initial apparition rates of released peptides varied according to the protein state (folded vs denatured) and informs the k off / k on ratio around the cleavage site. On the other hand, the time of appearance of a given peptide is related to its solvent accessibility and to the resilience of the residual protein structure in solution. Temperature-dependent digestion experiments allowed estimation of the type of secondary structures around the cleavage site.

Quality control of biopharmaceuticals such as monoclonal antibodies (mAbs) has been evolving and becoming more challenging as the requirements of the regulatory agencies increase due to the demanding complexity of products under evaluation. Mass Spectrometry (MS)-based methods such as the multi-attribute method (MAM) are being explored to achieve a deeper understanding of the attributes critical for the safety, efficacy, and quality of these products. MAM uses high mass accuracy/high-resolution MS data that enables the direct and simultaneous monitoring of relevant product quality attributes (PQAs, in particular, chemical modifications) in a single workflow, replacing several orthogonal methods, reducing time and costs associated with these assays. Here we describe a MAM implementation process using a QTOF high resolution platform. Method implementation was accomplished using NIST (National Institute for Standards and Technology) mAb reference material and an in-process mAb sample. PQAs as glycosylation profiles, methionine oxidation, tryptophan dioxidation, asparagine deamidation, pyro-Glu at N-terminal and glycation were monitored. Focusing on applications that require batch analysis and high-throughput, sample preparation and LC-MS parameters troubleshooting are discussed. This MAM workflow was successfully explored as reference analytical tool for comprehensive characterization of a downstream processing (DSP) polishing platform and for a comparability study following technology transfer between different laboratories., Competing Interests: Sanofi is also sponsor of this work. Anja Pfenninger, Martina Fischer, Michaela Strotbek and Nihal Tugçu are Sanofi employees and may hold shares and/or stock options in the company. The work described in this manuscript does not relate to any patients data, products in development or marketed products. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The relevant data to understand and replicate the method implementation reported in this work are within the manuscript and its Supporting Information files. Data containing antibody sequence (owned by Sanofi) cannot be shared publicly due to legal restrictions.

Doping quantum dots (QDs) with extra element presents a promising future for their applications in the fields of environmental monitoring, commercial products and biomedical sciences. However, it remains unknown for the influence of doping on the molecular biocompatibility of QDs and the underlying mechanisms of the interaction between doped-QDs and protein molecules. Using the "one-pot" method, we synthesized N-acetyl-l-cysteine capped CdTe: Zn 2+ QDs with higher fluorescence quantum yield, improved stability and better molecular biocompatibility compared with undoped CdTe QDs. Using digestive enzyme trypsin (TRY) as the protein model, the interactions of undoped QDs and Zn-doped QDs with TRY as well as the underlying mechanisms were investigated using multi-spectroscopy, isothermal titration calorimetry and dialysis techniques. Van der Waals forces and hydrogen bonds are the major driving forces in the interaction of both QDs with TRY, which leading to the loosening of protein skeleton and tertiary structural changes. Compared with undoped QDs, Zn-doped QDs bind less amount of TRY with a higher affinity and then release higher amount of Cd. Zn-doped QDs have a less stimulating impact on TRY activity by decreasing TRY binding and reducing Cd binding to TRY. Taken all together, Zn-doped QDs offer a safer alternative for the applications of QDs by reducing unwanted interactions with proteins and improving biocompatibility at the molecular level., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The cPEPmatch approach is a rapid computational methodology for the rational design of cyclic peptides to target desired regions of protein-protein interfaces. The method selects cyclic peptides that structurally match backbone structures of short segments at a protein-protein interface. In a second step, the cyclic peptides act as templates for designed binders by adapting the amino acid side chains to the side chains found in the target complex. A link to access the different tools that comprise the cPEPmatch method and a detailed step-by-step guide is provided. We outline the protocol by following the application to a trypsin protease in complex with the bovine inhibitor protein (BPTI). An extension of our original approach is also presented, where we give a detailed description of the usage of the cPEPmatch methodology focusing on identifying hot regions of protein-protein interfaces prior to the matching. This extension allows one to reduce the amount of evaluated putative cyclic peptides and to specifically design only those that compete with the strongest protein-protein binding regions. It is illustrated by an application to an MHC class I protein complex., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The poor stability of peptides against trypsin largely limits their development as potential antibacterial agents. Here, to obtain a peptide with increased trypsin stability and potent antibacterial activity, TICbf-14 derived from the cationic peptide Cbf-14 was designed by the addition of disulfide-bridged hendecapeptide (CWTKSIPPKPC) loop. Subsequently, the trypsin stability and antimicrobial and antibiofilm activities of this peptide were evaluated. The possible mechanisms underlying its mode of action were also clarified. The results showed that TICbf-14 exhibited elevated trypsin inhibitory activity and effectively mitigated lung histopathological damage in bacteria-infected mice by reducing the bacterial counts, further inhibiting the systemic dissemination of bacteria and host inflammation. Additionally, TICbf-14 significantly repressed bacterial swimming motility and notably inhibited biofilm formation. Considering the mode of action, we observed that TICbf-14 exhibited a potent membrane-disruptive mechanism, which was attributable to its destructive effect on ionic bridges between divalent cations and LPS of the bacterial membrane. Overall, TICbf-14, a bifunctional peptide with both antimicrobial and trypsin inhibitory activity, is highly likely to become an ideal candidate for drug development against bacteria., (© 2022. The Microbiological Society of Korea.)

Peptide mapping is a routine procedure for protein characterization in proteomics. This bottom-up analysis requires digestion of proteins into peptides before liquid chromatography- or capillary zone electrophoresis-mass spectrometry (LC-MS or CZE-MS, respectively). Proteins are usually digested off-line using proteolytic enzymes, typically trypsin, in solution or immobilized on appropriate supports. As an alternative, here we describe on-line immobilized enzyme microreactor capillary zone electrophoresis-mass spectrometry (IMER-CZE-MS) for a straightforward, rapid, and efficient protein digestion followed by separation, detection, and characterization of the generated peptides., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Sulfur mustard reacts with blood proteins, such as hemoglobin, to form stable adducts that can be used as long-lived biomarkers of exposure. These adducts can be analyzed by liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS) after an enzymatic digestion step. The objective of this study was to develop trypsin-based immobilized enzyme reactors (IMERs) in order to obtain a faster digestion of hemoglobin than the conventional in-solution digestion. Trypsin IMERs were synthetized by grafting the enzyme on a CNBr-Sepharose gel and the influence of several parameters on the digestion yields, such as the transfer volume between the injection loop and the IMER, the temperature and the digestion time was studied. The repeatability of the digestion on three laboratory-made IMERs was demonstrated for pure hemoglobin and hemoglobin previously exposed to different concentrations of sulfur mustard (RSD inferior to 13% and 21% respectively) and was better than that obtained for in-solution digestions (RSD inferior to 28% and up to 53% respectively). A preferential adduction of sulfur mustard on the histidine residues of hemoglobin was confirmed, for both in-solution and IMER digestion results. On a quantitative point of view, the performances of in-solution and IMER digestions were similar, with the theoretical possibility to detect peptides resulting from the in vitro incubation of hemoglobin in pure water with sulfur mustard at 7.5 ng⋅mL -1 . However, digestion on IMER proved to be more repeatable and 32 times faster than in-solution digestion, and a given IMER could be reused at least 60 times., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Trypsin is playing an important role in the processes of cancer proliferation, invasion and metastasis which require the precise information of morphology and mechanical properties on the nano-scale for the related research. In this work, living human hepatoma (SMCC-7721) cells were treated with different concentrations of trypsin solution. The morphology and mechanical properties of the cells were measured via atomic force microscope (AFM). Statistical analyses of measurement data indicated that with the increase of trypsin concentration, the average cell height and the surface roughness were both increased, but the cell viability, the cell surface adhesion and the elasticity modulus were decreased significantly. The force required to puncture the cells was also gradually reduced. It indicates that trypsin not only hydrolyses the proteins between the cell and the substrate but also the membrane proteins. The results offer valuable clues for the cancerous process study, pathological analysis and trypsin inhibitor drug development. And this work provides an effective way for overcoming the cell membrane in drug injection for cell-targeted therapy., (© 2021 Royal Microscopical Society.)

Molecular imprinting represents one of the most promising strategies to design artificial enzyme inhibitors. However, the study of molecularly imprinted enzyme inhibitors (MIEIs) remains at a primary stage. Advanced applications of MIEIs for cell regulation have rarely been explored. Using a solid-phase oriented imprinting strategy so as to leave the active site of the enzymes accessible, we synthesized two MIEIs that exhibit high specificity and potent inhibitory effects (inhibition constant at low nM range) towards trypsin and angiogenin. The trypsin MIEI inhibits trypsin activity, tryptic digestion-induced extracellular matrix lysis and cell membrane destruction, indicating its utility in the treatment of active trypsin-dependent cell injury. The angiogenin MIEI blocks cancer cell proliferation by suppressing the ribonuclease activity of angiogenin and decreasing the angiogenin level inside and outside HeLa cells. Our work demonstrates the versatility of MIEIs for both enzyme inhibition and cell fate manipulation, showing their great potential as therapeutic drugs in biomedicine., (© 2021 Wiley-VCH GmbH.)

Nuclear spin relaxation dispersion parameters are proposed as indicators of the binding mode of a ligand to a protein. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) provided a 13 C signal enhancement between 3000-6000 for the ligand 4-(trifluoromethyl) benzene-1-carboximidamide binding to trypsin. The measurement of 13 C R 2 relaxation dispersion was enabled without isotope enrichment, using a series of single-scan Carr-Purcell-Meiboom-Gill experiments with variable refocusing delays. The magnitude in dispersion for the spins of the ligand is correlated to the position with respect to the salt bridge between protein and the amidine group of the ligand, indicating the ligand binding orientation. Hyperpolarized relaxation dispersion is an alternative to chemical shift or NOE measurements for determining ligand binding modes., (© 2021 Wiley-VCH GmbH.)

Complete characterization and quantification of monoclonal antibodies often rely on enzymatic digestion with trypsin. In order to accelerate and automate this frequently performed sample preparation step, immobilized enzyme reactors (IMER) compatible with standard HPLC systems were used. This allows an automated online approach in all analytical laboratories. We were able to demonstrate that the required digestion time for the model monoclonal antibody rituximab could be reduced to 20 min. Nevertheless, a previous denaturation of the protein is required, which also needs 20 min. Recoveries were determined at various concentrations and were 100% ± 1% at 100 ng on column, 96% ± 7% at 250 ng on column and 98% ± 2% at 450 ng on column. Despite these good recoveries, complete digestion was not achieved, resulting in a poorer limit of quantification. This is 50 ng on column under optimized IMER conditions, whereas an offline digest on the same system achieved 0.3 ng on column. Furthermore, our work revealed that TRIS buffers, when used with an IMER system, led to alteration of the peptides and induced modifications in the peptides. Therefore, the addition of TRIS should be avoided when working at elevated temperatures of about 60 °C. Nevertheless, our results have shown that the recovery is not significantly influenced whether TRIS is used or not (recovery: 96 ± 7% with TRIS vs. 100 ± 9% without TRIS)., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.