ABSTRACT
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.
Subject(s)
Hydrogels , Pancreatic Fistula , Peptides , Pancreatic Fistula/prevention & control , Animals , Rats , Hydrogels/chemistry , Male , Peptides/pharmacology , Peptides/chemistry , Peptides/metabolism , Chymotrypsin/metabolism , Postoperative Complications/prevention & control , Trypsin/metabolism , Trypsin/chemistry , Lipase/metabolism , Humans , Rats, Sprague-Dawley , Disease Models, Animal , Pancreas/enzymology , Pancreas/pathologyABSTRACT
Gas cluster ion beam (GCIB)-assisted deposition is used to build multilayered protein-based structures. In this process, Ar3000-5000+ clusters bombard and sputter molecules from a reservoir (target) to a collector, an operation that can be sequentially repeated with multiple targets. The process occurs under a vacuum, making it adequate for further sample conservation in the dry state, since many proteins do not have long-term storage stability in the aqueous state. First of all, the stability in time and versatility in terms of molecule selection are demonstrated with the fabrication of peptide multilayers featuring a clear separation. Then, lysozyme and trypsin are used as protein models to show that the activity remaining on the collector after deposition is linearly proportional to the argon ion dose. The energy per atom (E/n) of the Ar clusters is a parameter that was also changed for lysozyme deposition, and its increase negatively affects activity. The intact detection of larger protein molecules by SDS-PAGE gel electrophoresis and a bioassay (trypsin at ≈25 kDa and glucose oxidase (GOx) at ≈80 kDa) is demonstrated. Finally, GOx and horseradish peroxidase, two proteins involved in the same enzymatic cascade, are successively deposited on ß-d-glucose to build an on-demand release material in which the enzymes and the substrate (ß-d-glucose) are combined in a dry trilayer, and the reaction occurs only upon reintroduction in aqueous medium.
Subject(s)
Glucose Oxidase , Horseradish Peroxidase , Muramidase , Trypsin , Muramidase/chemistry , Muramidase/metabolism , Glucose Oxidase/chemistry , Glucose Oxidase/metabolism , Trypsin/chemistry , Trypsin/metabolism , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , Peptides/chemistry , Animals , Glucose/chemistryABSTRACT
The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6's large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde's spacing arm, reducing spatial hindrance and enhancing enzyme-substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.
Subject(s)
Acrylamide , Dopamine , Enzymes, Immobilized , Polyethyleneimine , Serum Albumin, Bovine , Trypsin , Polyethyleneimine/chemistry , Dopamine/chemistry , Dopamine/metabolism , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Acrylamide/chemistry , Trypsin/chemistry , Trypsin/metabolism , Animals , Cattle , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism , Porosity , Hydrophobic and Hydrophilic Interactions , Hemoglobins/chemistry , Hemoglobins/metabolism , ProteolysisABSTRACT
Developing strategies to target injured pancreatic acinar cells (PACs) in conjunction with primary pathophysiology-specific pharmacological therapy presents a challenge in the management of acute pancreatitis (AP). We designed and synthesized a trypsin-cleavable organosilica precursor bridged by arginine-based amide bonds, leveraging trypsin's ability to selectively identify guanidino groups on arginine via Asp189 at the active S1 pocket and cleave the carboxy-terminal (C-terminal) amide bond via catalytic triads. The precursors were incorporated into the framework of mesoporous silica nanoparticles (MSNs) for encapsulating the membrane-permeable Ca2+ chelator BAPTA-AM with a high loading content (â¼43.9%). Mesenchymal stem cell membrane coating and surface modification with PAC-targeting ligands endow MSNs with inflammation recruitment and precise PAC-targeting abilities, resulting in the highest distribution at 3 h in the pancreas with 4.7-fold more accumulation than that of naked MSNs. The outcomes transpired as follows: After bioinspired MSNs' skeleton biodegradation by prematurely and massively activated trypsin, BAPTA-AM was on-demand released in injured PACs, thereby effectively eliminating intracellular calcium overload (reduced Ca2+ level by 81.3%), restoring cellular redox status, blocking inflammatory cascades, and inhibiting cell necrosis by impeding the IκBα/NF-κB/TNF-α/IL-6 and CaMK-II/p-RIP3/p-MLKL/caspase-8,9 signaling pathways. In AP mice, a single dose of the formulation significantly restored pancreatic function (lipase and amylase reduced more by 60%) and improved the survival rate from 50 to 91.6%. The formulation offers a potentially effective strategy for clinical translation in AP treatment.
Subject(s)
Pancreatitis , Trypsin , Animals , Pancreatitis/drug therapy , Pancreatitis/pathology , Pancreatitis/metabolism , Trypsin/metabolism , Trypsin/chemistry , Mice , Porosity , Nanomedicine , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Organosilicon Compounds/chemistry , Organosilicon Compounds/pharmacology , Male , Humans , Acinar Cells/drug effects , Acinar Cells/metabolism , Acinar Cells/pathology , Mice, Inbred C57BLABSTRACT
The development of a green and facile method for the controlled synthesis of functional polypeptides is desired for sustainable material applications. In this study, the regioselective synthesis of poly(l-lysine) (polyLys) via enzyme-catalyzed aminolysis was achieved by bulk polymerization of l-lysine ethyl ester (Lys-OEt) using immobilized Candida antarctica lipase Novozym 435 (IM-lipase) or trypsin (IM-trypsin). Structural characterization of the obtained polyLys revealed that IM-lipase resulted solely in ε-linked amide bond formation, whereas IM-trypsin predominantly provided α-linked polyLys. Optimization of the conditions for the bulk polymerization using immobilized enzymes resulted in high monomer conversion and a high degree of polymerization, with excellent regioselectivity. Molecular docking simulations revealed different binding conformations of Lys-OEt to the catalytic pockets of lipase and trypsin, which putatively resulted in different amino moieties being used for amide bond formation. The immobilized enzymes were recovered and recycled for bulk polymerization, and the initial activity was maintained in the case of IM-trypsin. The obtained α- and ε-linked polyLys products exhibited different degradability against proteolysis, demonstrating the possibility of versatile applications as sustainable materials. This enzymatic regioregular control enabled the synthesis of well-defined polypeptide-based materials with a diverging structural variety.
Subject(s)
Enzymes, Immobilized , Fungal Proteins , Lipase , Polymerization , Trypsin , Lipase/chemistry , Lipase/metabolism , Enzymes, Immobilized/chemistry , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Trypsin/chemistry , Trypsin/metabolism , Polylysine/chemistry , Lysine/chemistry , Molecular Docking Simulation , Biocatalysis , Esters/chemistry , BasidiomycotaABSTRACT
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.
Subject(s)
Collagenases , Intervertebral Disc , Trypsin , Animals , Cattle , Trypsin/metabolism , Intervertebral Disc/metabolism , Collagenases/metabolism , Biomechanical Phenomena , Mechanical Phenomena , Injections , Pressure , Nucleus Pulposus/metabolismABSTRACT
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.
Subject(s)
Ceruletide , Chymotrypsin , Mice, Inbred C57BL , Mice, Knockout , Pancreatitis , Trypsin , Animals , Chymotrypsin/metabolism , Chymotrypsin/genetics , Ceruletide/toxicity , Pancreatitis/chemically induced , Pancreatitis/pathology , Pancreatitis/metabolism , Pancreatitis/genetics , Mice , Trypsin/metabolism , Secretagogues/metabolism , Pancreas/metabolism , Pancreas/pathology , Disease Models, Animal , MaleABSTRACT
Cyclic peptides (CPs) are emerging as promising drug candidates. Numerous natural CPs and their analogs are effective therapeutics against various diseases. Notably, many of them contain peptidyl cis-prolyl bonds. Due to the high rotational barrier of peptide bonds, conventional molecular dynamics simulations struggle to effectively sample the cis/trans-isomerization of peptide bonds. Previous studies have highlighted the high accuracy of the residue-specific force field (RSFF) and the high sampling efficiency of high-temperature molecular dynamics (high-T MD). Herein, we propose a protocol that combines high-T MD with RSFF2C and a recently developed reweighting method based on probability densities for accurate structure prediction of proline-containing CPs. Our method successfully predicted 19 out of 23 CPs with the backbone rmsd < 1.0 Å compared to X-ray structures. Furthermore, we performed high-T MD and density reweighting on the sunflower trypsin inhibitor (SFTI-1)/trypsin complex to demonstrate its applicability in studying CP-complexes containing cis-prolines. Our results show that the conformation of SFTI-1 in aqueous solution is consistent with its bound conformation, potentially facilitating its binding.
Subject(s)
Molecular Dynamics Simulation , Peptides, Cyclic , Proline , Peptides, Cyclic/chemistry , Proline/chemistry , Trypsin/chemistry , Trypsin/metabolism , Temperature , Protein ConformationABSTRACT
Serine proteases (SPs) are distributed among all living cells accounting for almost one-third of all proteases. Dysregulation of SPs during inflammation and/or infection can result in devastating consequences, such as skin and lung inflammation, neuroinflammation, arthritis, as well as metastasis of cancerous cells. Such activities are tightly regulated by various inhibitors known as serine protease inhibitors (SERPIN). The thermodynamic investigations previously revealed that L-ascorbic acid binds to trypsin more firmly than pepsin and the binding force of L-ascorbic acid is driven by hydrogen bonds and van der Waals forces. However, the physiochemical effects of such interaction on trypsin and/or pepsin have not yet been reported. Ascorbic acid, also known as vitamin C, is one of the essential nutrients and most common food supplements, fortificants, and preservatives. The aim of this study was to explore the inhibitory effects of ascorbic acid on serine proteases at various concentrations on the in-vitro digestion and/or hydrolysis of intercellular matrix of cell monolayer and human serum albumin (HSA). The inhibitory effects of ascorbic on trypsin are investigated by qualitative and quantitative analysis using SDS-PAGE imaging and NIH densitometric software. Upon the addition of ascorbic acid in both indicator systems, the detachment and/or dissociation of cell monolayer and the digestion of HSA were inhibited in the presence of EDTA-Trypsin. The inhibitory effect of ascorbic acid on the digestion of intercellular matrix and/or hydrolysis of HSA showed a dose-dependent trend until it reached the maximum extent of inhibition. At an equal concentration (2.5mg/mL) ascorbic acid and EDTA-Trypsin exhibited the most potent inhibitory effect on the in vitro digestion of protein either in the form of intercellular matrix in cell monolayer and/or HSA respectively. Overall, our results based on two indicator systems strongly indicate that ascorbic acid may function as a serine protease inhibitor (SERPIN) beyond other important functions.
Subject(s)
Ascorbic Acid , Serine Proteinase Inhibitors , Humans , Ascorbic Acid/pharmacology , Ascorbic Acid/chemistry , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Serum Albumin, Human/metabolism , Serum Albumin, Human/chemistry , Trypsin/metabolism , Trypsin/chemistry , Cell Line, Tumor , A549 CellsABSTRACT
An electrophilic arginine mimetic, 2-chloroacetamidine (CAM), was deployed to enable trypsin-mediated proteolysis at cysteine residues and to enhance mass spectrometry-based proteomic detection of cysteine-containing peptides. Illustrating the value of the CAM-capping strategy, proteogenomic analysis using a two-stage false discovery rate (FDR) search revealed >50% enhanced coverage of missense variants, when compared to established workflows.
Subject(s)
Cysteine , Trypsin , Cysteine/chemistry , Trypsin/metabolism , Trypsin/chemistry , Acetamides/chemistry , Proteolysis , Proteomics , Peptides/chemistry , HumansABSTRACT
Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.
Subject(s)
Proteomics , Trypsin , Trypsin/metabolism , Trypsin/chemistry , Proteomics/methods , Isotope Labeling/methods , Filtration , Peptide Fragments/analysis , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Mass Spectrometry/methods , Humans , Hydrogen-Ion Concentration , Animals , Peptides/chemistry , Peptides/analysisABSTRACT
Quantitative proteomics approaches based on stable isotopic labeling and mass spectrometry have been widely applied to disease research, drug target discovery, biomarker identification, and systems biology. One of the notable stable isotopic labeling approaches is trypsin-catalyzed 18O/16O labeling, which has its own advantages of low sample consumption, simple labeling procedure, cost-effectiveness, and absence of chemical reactions that potentially generate by-products. In this chapter, a protocol for 18O/16O labeling-based quantitative proteomics approach is described with an application to the identification of proteomic biomarkers of acetaminophen (APAP)-induced hepatotoxicity in rats. The protocol involves first the extraction of proteins from liver tissues of control and APAP-treated rats and digestion into peptides by trypsin. After cleaning of the peptides by solid-phase extraction, equal amounts of peptides from the APAP treatment and the control groups are then subject to trypsin-catalyzed 18O/16O labeling. The labeled peptides are combined and fractionated by off-line strong cation exchange liquid chromatography (SCXLC), and each fraction is then analyzed by nanoflow reversed-phase LC coupled online with tandem mass spectrometry (RPLC-MS/MS) for identification and quantification of differential protein expression between APAP-treated rats and controls. The protocol is applicable to quantitative proteomic analysis for a variety of biological samples.
Subject(s)
Acetaminophen , Biomarkers , Chemical and Drug Induced Liver Injury , Isotope Labeling , Liver , Proteomics , Tandem Mass Spectrometry , Acetaminophen/toxicity , Acetaminophen/adverse effects , Isotope Labeling/methods , Proteomics/methods , Animals , Rats , Biomarkers/metabolism , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Chemical and Drug Induced Liver Injury/etiology , Tandem Mass Spectrometry/methods , Liver/metabolism , Liver/drug effects , Liver/pathology , Proteome/metabolism , Proteome/analysis , Trypsin/metabolism , Oxygen Isotopes/metabolismABSTRACT
Abamectin has been extensively used in paddy fields to control insect pests. However, little information is available regarding its effects on non-target insects. In this study, we performed acute (3rd instar larvae) and chronic toxicity (newly hatched larvae <24 h) to determine the toxicity effects of abamectin on Chironomus kiiensis. The median lethal concentration (LC50) values of 24 h and 10 d were 0.57 mg/L and 68.12 µg/L, respectively. The chronic exposure significantly prolonged the larvae growth duration and inhibited pupation and emergence. The transcriptome and biochemical parameters were measured using 3rd instar larvae exposed to acute LC10 and LC25 for 24 h. Transcriptome data indicated that five trypsin and four chymotrypsin genes were downregulated, and RT-qPCR verified a significant expression decrease in trypsin3 and chymotrypsin1 genes. Meanwhile, abamectin could significantly inhibit the activities of the serine proteases trypsin and chymotrypsin. RNA interference showed that silencing trypsin3 and chymotrypsin1 genes led to higher mortality of C. kiiensis to abamectin. In conclusion, these findings indicated that trypsin and chymotrypsin are involved in the abamectin toxicity against C. kiiensis, which provides new insights into the mechanism of abamectin-induced ecotoxicity to chironomids.
Subject(s)
Chironomidae , Chymotrypsin , Ivermectin , Larva , Trypsin , Animals , Chymotrypsin/metabolism , Chymotrypsin/genetics , Chironomidae/drug effects , Chironomidae/genetics , Trypsin/metabolism , Trypsin/genetics , Ivermectin/analogs & derivatives , Ivermectin/toxicity , Larva/drug effects , Insecticides/toxicityABSTRACT
Trypsin digestion plays a pivotal role in successful bottom-up peptide characterization and quantitation. While denaturants are often incorporated to enhance protein solubility, surfactants are recognized to inhibit enzyme activity. However, several reports have suggested that incorporating surfactants or other solvent additives may enhance digestion and MS detection. Here, we assess the impacts of ionic surfactants on cumulative trypsin activity and subsequently evaluate the total digestion efficiency of a proteome mixture by quantitative MS. Although low surfactant concentrations, such as 0.01% SDS or 0.2% SDC, significantly enhanced the initial trypsin activity (by 14 or 42%, respectively), time course assays revealed accelerated enzyme deactivation, evident by 10- or 40-fold reductions in trypsin activity half-life at these respective surfactant concentrations. Despite enhanced initial tryptic activity, quantitative MS analysis of a common liver proteome extract, digested with various surfactants (0.01 or 0.1% SDS, 0.5% SDC), consistently revealed decreased peptide counts and signal intensity, indicative of a lower digestion efficiency compared to a nonsurfactant control. Furthermore, including detergents for digestion did not improve the detection of membrane proteins, nor hydrophobic peptides. These results stress the importance of assessing cumulative enzyme activity when optimizing the digestion of a proteome mixture, particularly in the presence of denaturants.
Subject(s)
Proteome , Proteomics , Surface-Active Agents , Trypsin , Trypsin/metabolism , Trypsin/chemistry , Surface-Active Agents/pharmacology , Surface-Active Agents/chemistry , Proteome/analysis , Proteomics/methods , Animals , Sodium Dodecyl Sulfate/pharmacology , Sodium Dodecyl Sulfate/chemistry , Liver/metabolism , Liver/enzymology , Liver/drug effectsABSTRACT
Glycation and the subsequent formation of advanced glycation end products (AGEs) disrupt and impair the physiological functions of proteins. This study presents a comprehensive glycation site mapping of human serum albumin (HSA) utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both in vitro glycation experiments and patient samples were investigated, exploring various enzymes, processing techniques, and their impacts on glycation site detection. A pilot study was conducted, analyzing sixteen serum samples, which spanned from healthy individuals to severe diabetic patients (with HbA1c values ranging from 5.7% to 18.1%). The aim was to comprehend the progression of glycation on various sites of HSA with increasing levels of glycation. Their glycated albumin levels (GA) spanned from 19.7% to 62.3%. Trypsin-mediated proteolytic digestion unveiled 12 glycation sites through direct in-solution digestion of whole serum. However, isolating albumin from serum enabled the identification of a higher number of glycation sites in each sample compared to direct serum digestion. Boronate affinity chromatography facilitated the segregation of less glycated albumin (LGA) from the more glycated albumin (MGA) fraction. Subsequent proteolytic digestion of both LGA and MGA samples revealed similar glycation sites. The MGA fraction exhibited a greater number of identified glycation sites, thereby elucidating which sites are particularly prone to glycation in highly glycated albumin samples. Changes in relative glycation levels were noted in the tryptic digests of albumin samples following the sample enrichment steps, as opposed to direct in-solution digestion of whole serum. Two enzymes, trypsin and Glu-C, were evaluated for efficacy in sequence coverage and glycation site analysis of HSA, with trypsin demonstrating superior efficiency over Glu-C.
Subject(s)
Glycated Serum Albumin , Glycation End Products, Advanced , Serum Albumin, Human , Tandem Mass Spectrometry , Humans , Glycation End Products, Advanced/chemistry , Tandem Mass Spectrometry/methods , Serum Albumin, Human/chemistry , Glycosylation , Chromatography, Liquid/methods , Serum Albumin/chemistry , Serum Albumin/analysis , Serum Albumin/metabolism , Pilot Projects , Diabetes Mellitus/blood , Trypsin/chemistry , Trypsin/metabolismABSTRACT
Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely αA- and γ-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.
Subject(s)
Epithelial Cells , Lens, Crystalline , Trypsin , Epithelial Cells/cytology , Lens, Crystalline/cytology , Animals , Mice , Trypsin/chemistry , Trypsin/metabolism , Cell Culture Techniques/methods , Primary Cell Culture/methodsABSTRACT
Development of an energy-driven self-assembly process is a matter of interest for understanding and mimicking diverse ranges of biological and environmental patterns in a synthetic system. In this article, first we demonstrate transient and temporally controlled self-assembly of a DNA-histone condensate where trypsin (already present in the system) hydrolyzes histone, resulting in disassembly. Upon performing this dynamic self-assembly process in a gel matrix under an electric field, we observe diverse kinds of DNA patterning across the gel matrix depending on the amount of trypsin, incubation time of the reaction mixture, and gel porosity. Notably, here, the micrometer-sized DNA-histone condensate does not move through the gel and only free DNA can pass; therefore, transport and accumulation of DNA at different zones depend on the release rate of DNA by trypsin. Furthermore, we show that the viscoelasticity of the native gel increases in the presence of DNA and a pattern over gel viscoelasticity at different zones can be achieved by tuning the amount of enzyme, i.e., the dissociation rate of the DNA-histone condensate. We believe enabling spatiotemporally controlled DNA patterning by applying an electric field will be potentially important in designing different kinds of spatiotemporally distinct dynamic materials.
Subject(s)
DNA , Elasticity , Histones , Hydrogels , Trypsin , DNA/chemistry , Histones/chemistry , Histones/metabolism , Trypsin/chemistry , Trypsin/metabolism , Hydrogels/chemistry , Viscosity , ElectrophoresisABSTRACT
The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.
Subject(s)
Peptides , Trypsin , Humans , Trypsin/chemistry , Trypsin/metabolism , Peptides/immunology , Peptides/chemistry , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Proteomics/methods , Ion Mobility Spectrometry/methodsABSTRACT
Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.
Subject(s)
Fibrinogen , Tandem Mass Spectrometry , Tyrosine , Tyrosine/chemistry , Tyrosine/analogs & derivatives , Tandem Mass Spectrometry/methods , Fibrinogen/chemistry , Fibrinogen/metabolism , Chromatography, Liquid/methods , Humans , Protein Processing, Post-Translational , Trypsin/chemistry , Trypsin/metabolism , Sulfates/chemistry , Amino Acid Sequence , Peptides/chemistry , Peptides/analysis , ElectronsABSTRACT
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 Ce4+ 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.