ANALYTICA CHIMICA ACTA
·
2017
·
IF 4.95
Yujia Wang,Shucheng Sun,Irfan Azhar,Qian Zhang,Qishu Qu,Li Yang
Core-shell mesoporous silica (CSMS) microspheres with tunable mesopores in the shell are highly desired in various bioapplications. With novel CSMS microspheres that are synthesized using a convenient two-phase process, we report in this study the analysis of low molecular-weight (MW < 30 kDa) proteins by combining size-exclusion separation and enzyme immobilization . The obtained CSMS microspheres possess uniform diameter (1.3 μm with a shell thickness of 57 nm), large and tunable perpendicular mesopores (7.9 nm), high surface area (55.5 m 2 /g), large pore volume (0.12 cm 3 /g) and excellent water dispersibility. The CSMS microsphere-based enzyme nanoreactors have been fabricated by immobilizing trypsin on the pore channels of the CSMS microspheres using either physical absorption or covalent binding via thiol or aldehyde group with a high loading capacity of 11.8–6.1 mg/g. Due to the unique fibrous pore structure , low MW proteins can enter the channels in the shell to interact with immobilized trypsin, followed by analysis of the digestion products using MALDI-TOF MS or electrophoresis (CE) techniques. The properties and analytical performance of different trypsin-immobilized CSMS microspheres has been systematically evaluated. The results show that the peptide-sequence coverage of the smaller protein is enhanced by using trypsin-CSMS microspheres, indicating the size-dependent digestion which results from the size-exclusion interaction of the mesopores against the high-MW proteins. The present study would pave the way for further applications of mesoporous materials in proteome analysis.
研究领域:
生物技术
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分析化学
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材料科学
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DOI
PubMed
高影响因子
TALANTA
·
2017
·
IF 4.162
Shanshan Huang,Fangfang Li,Caiyun Liao,Baozhan Zheng,Juan Du,Dan Xiao
A simple fluorescent probe HBI-GR based on the combination of the fluorophore (p-HBI) in green fluorescent protein (GFP) and Guanine riboside (GR) for HSA was successfully synthesized. HBI-GR showed an obvious fluorescence enhancement toward HSA without interference from other proteins, amino acids, anions and commonly existing metal ions . HBI-GR exhibited high sensitivity towards HSA with a good linear relationship between the fluorescence intensity of HBI-GR and HSA concentration from 0 to 0.06 mg mL −1 . The limit of detection, based on a signal-to-noise ratio of 3, was 15.09 ng mL −1 , which was much lower than that of most other reported probes. HBI-GR was almost non-fluorescent because of the bond twisting in the exited state of chromophore HBI. After binding to the hydrophobic pocket of HSA, it showed an obvious fluorescence enhancement due to the rigidifying of the flexible chromophore HBI by the hydrophobic environment. The resulting HBI-GR/HSA system also showed a satisfactory sensing ability toward trypsin through decreased fluorescence intensity with the detection limit of 0.0282 ng mL −1 . The fluorescence decreasing process was occurred as the lysine and arginine amino acids residues of HSA were cleaved by trypsin, which led to further exposure of HBI-GR to the PBS buffer phase and a concomitant decrease of the HBI-GR fluorescence intensity. Moreover, the probe HBI-GR was successfully used to detect HSA in healthy human urine and human blood serum samples. The practical application of the HBI-GR/HSA system for trypsin detection in healthy human urine also achieved satisfactory result.
研究领域:
生物化学
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分析化学
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PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2017
·Vol. 409
·期 8
·2179-2187
·
IF 3.431
Wang Heping,Jiao Fenglong,Gao Fangyuan,Zhao Xinyuan,Zhao Yan,Shen Yehua,Zhang Yangjun,Qian Xiaohong
Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268 μg mg −1 ). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2 min. For the digestion of proteins extracted from Amygdalus pedunculata , a total of 2833 protein groups were identified, which was slightly more than those obtained by 12 h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis.
Covalent organic frameworks-coated magnetic graphene was prepared as novel carrier for highly efficient tryptic immobilization
研究领域:
生物化学
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蛋白质组学
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材料科学
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PubMed
高影响因子
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2015
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IF 2.353
Yingying Liu,Guowen Zhang,Yijing Liao,Yaping Wang
Psoralen (PSO) is a naturally occurring furanocoumarin with a variety of pharmacological activities, however very limited information on the interaction of PSO with trypsin is available. In this study, the binding characteristics between PSO and trypsin at physiological pH were investigated using a combination of fluorescence, UV–vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopic, chemometric and molecular modeling approaches. It was found that the fluorescence quenching of trypsin by PSO was a static quenching procedure, ascribing the formation of a PSO–trypsin complex. The binding of PSO to trypsin was driven mainly by hydrophobic forces as the positive enthalpy change and entropy change values. The molecular docking showed that PSO inserted into the active site pocket of trypsin to interact with the catalytic residues His57, Asp102 and Ser195 and may cause a decrease in trypsin activity. The results of CD and FT-IR spectra along with the temperature-induced denaturation studies indicated that the addition of PSO to trypsin led to the changes in the secondary structure of the enzyme. The concentration profiles and spectra of the three components (PSO, trypsin, and PSO–trypsin complex) obtained by multivariate curve resolution-alternating least squares analysis exhibited the kinetic processes of PSO–trypsin interaction. This study will be helpful to understand the mechanism of PSO that affects the conformation and activity of trypsin in biological processes.
研究领域:
生物化学
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药理学
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分子生物学
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2014
·
IF 3.107
Yan-Qing Wang,Hong-Mei Zhang
The effects of bisphenol S on the structures and activities of trypsin and pepsin were investigated by various methods like UV–visible absorbance, fluorescence, circular dichroism, and molecular docking. The secondary and tertiary structures of trypsin and pepsin were altered by bisphenol S binding, which resulted in the loosening of the skeletons of trypsin and pepsin. In addition, bisphenol S induced microenvironmental changes around tyrosine and tryptophan residues of trypsin and pepsin. The activity experimental results showed that the activity of pepsin decreases obviously with the increasing concentration of BPS, while the activity of trypsin does not change remarkably. The binding and thermodynamic parameters obtained by molecular docking and fluorescence spectroscopy showed that the bindings of bisphenol S to trypsin and pepsin were spontaneous processes and hydrogen bonding and hydrophobic interactions played a vital role in stabilizing the bisphenol S–trypsin and bisphenol S–pepsin complexes. The binding constants (KA) of bisphenol S with trypsin were 7.42 × 104 (298 K) and 5.91 × 104 L/mol (310 K), and those of pepsin were 5.78 × 104 (298 K) and 4.44 × 104 L/mol (310 K). Moreover, there was one main kind of binding site for bisphenol S on trypsin or pepsin.
研究领域:
生物化学
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环境毒理学
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DOI
PubMed
高影响因子
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2013
·
IF 1.977
Hua Li,Juanjuan Pu,Yi Wang,Chuang Liu,Jie Yu,Tao Li,Ruiqiang Wang
The interactions between Trypsin and bifendate (DDB) or analogs (I, II and III) were investigated by fluorescence, UV–visible absorption, resonance light scattering, synchronous fluorescence and 3D spectroscopy under mimic physiological conditions. The results revealed that DDB and analogs caused the fluorescence quenching of Trypsin by the formation of DDB/I/II/III–Trypsin complex. The quenching and energy transfer mechanisms were discussed. The binding constants and thermodynamic parameters at three different temperatures were obtained. The hydrophobic interaction was the predominant intermolecular forces to stabilize the complex. Results showed that DDB was the stronger quencher and bound to Trypsin with higher affinity than other three analogs.
研究领域:
生物化学
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药物化学
·
分子生物学
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2023
·Vol. 190
·期 4
·1-8
·
IF 5.7
Wu Ning-Ning,Chen Lin-Ge,Xiao Min-Zhi,Yuan Rong-Yao,Wang Hai-Bo
A fluorescent method is described for trypsin determination through the strong electrostatic interactions between cationic polyelectrolytes and single-stranded DNA (ssDNA) templated Au nanoclusters (AuNCs). The ssDNA-AuNCs display improved fluorescence emission with excitation/emission maxima at 280/475 nm after being incorporated with poly(diallyldimethylammonium chloride) (PDDA). Fluorescent enhancement is mainly attributed to the electrostatic interactions occurring between PDDA and ssDNA templates. This can make the conformation of the ssDNA templates to change. Thus, it offers a better microenvironment for stabilizing and protecting ssDNA-AuNCs, and results in fluorescence emission enhancement. By using protamine as a model, the method is employed for the determination of trypsin. The assay enables trypsin to be determined with good sensitivity and a linear response ranging from 5 ng⋅mL −1 to 60 ng⋅mL −1 with a 1.5 ng⋅mL −1 limit of detection. It is also extended to determine the trypsin contents in human’s serum samples with recoveries between 98.7% and 103.5% with relative standard deviations (RSDs) between 3.5% and 4.8%. Graphical abstract A novel fluorescent strategy has been developed for of trypsin determination by using protamine mediated fluorescent enhancement of DNA templated Au nanoclusters.
研究领域:
分析化学
·
生物化学
·
纳米技术
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PubMed
高影响因子
ACS Omega
·
2017
·
IF 0
Jie Feng,Yonglei Chen,Yangxia Han,Juanjuan Liu,Sudai Ma,Huige Zhang,Xingguo Chen
In this article, a simple protocol to prepare water-soluble fluorescent copper nanoclusters (CuNCs) using trypsin as a stabilizer and hydrazine hydrate as a reducing agent was reported. It was found that the pH of the reaction solution was critical in determining the fluorescence of CuNCs. CuNCs with blue and yellow fluorescent emission were obtained under basic and acidic conditions, respectively. Although the detailed formation mechanisms of these CuNCs required further analysis, the synthetic route was promising for preparing different fluorescent metal NCs for applications. With good water solubility and excellent photostability, the yellow-emitting CuNCs could serve as a fluorescence probe for detection of Hg2+ based on the aggregation-induced quenching mechanism. The fluorescence quenching efficiency had fantastic linearity to Hg2+ concentrations in the range of 0.1–100 μM, with a limit of detection of 30 nM. Additionally, the yellow-emitting CuNCs exhibited negligible cytotoxicity and were successfully applied to bioimaging of HeLa cells.
研究领域:
纳米技术
·
分析化学
·
生物医学工程
·
材料科学
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DOI
PubMed
SYNTHETIC COMMUNICATIONS
·
2024
·
IF 1.8
Xin-Hao Hua,Fa-Qi Wang,Zhong-Jin Yang,Yuan Liu,Shu-Han Yang,Dong-Rong Zhu,Yu-Ming Liu
Herein, we designed a series of guanidinobenzoic acid ester derivatives on the basis of approved AP drugs, such as nafamostat, gabexate and camostat, and evaluated their inhibitory effects on trypsin and anti-inflammatory activity. Among them, five compounds (6a, 6c–6e, 7j) showed excellent inhibitory effects on trypsin with IC50 values of 0.0756 μM to 0.1227 μM, which are more potent than nafamostat and gabexate. Moreover, compounds 6a, 6b and 6c also showed significant inhibitory potency against the pro-inflammatory molecule NO with IC50 values of 1.618 μM, 2.276 μM, and 3.022 μM, respectively. Consequently, the potential lead compounds simultaneously with anti-trypsin and anti-inflammatory activities were identified, which would profit further structural optimization for the treatment of AP.
研究领域:
药物化学
·
炎症研究
·
消化系统疾病
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DOI
高影响因子
ACS Nano
·
2024
·
IF 15.8
Yanan Wang,Deyao Qian,Xinyuan Wang,Xue Zhang,Zerui Li,Xinlei Meng,Liangmin Yu,Xuefeng Yan,Zhiyu He
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 (∼43.9%). 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.
研究领域:
纳米医学
·
药学
·
生物医学工程
·
分子生物学
·
炎症研究
·
药物递送系统
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PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
·
2025
·Vol. 23
·期 1
·1-20
·
IF 12.6
Wu Can,Zhang Xiao Wei,Wang Manman,Sun Jinpan,Chen Jianfei,Guan Yanbin,Pang Xin
Chemotherapy remains an effective treatment for colon cancer but is hampered by its limited response rate. Bioactive peptides, marked with intracellular transformations, have been reported as an effective approach to boosting chemotherapeutic activity. Herein, a promising trypsin-responsive bioactive peptide-based nanodrug is constructed, which could significantly prolong the drug retention time in vivo by cascading transformations and improve chemotherapeutic efficacy. Initially, 1-Pept co-assembles with Dox into a few nanofibers called 1-Pept/Dox NFs, inducing an enhanced cellular uptake via caveolae-mediated endocytosis by avoiding lysosomal degradation and further promoting perinuclear transportation, thus enlarging the drug efficacy in target areas. After nanofiber disassembly, the released 1-Pept converts into Pept under the catalysis of intracellular overexpressed trypsin, which then reassembles into denser Pept NFs, inducing a cascade of effects including disruption of the cytoskeleton, mitochondrial dysfunction, and activation of caspase-3. By the synergism of Pept NFs and Dox, caspase-3 can be further activated, and cause greater damage to nuclear, thereby leading to tumor ablation. As the first example of employing trypsin-mediated nanodrugs with cascading transformations to promote chemotherapeutic activity, this work promises a strategy for novel therapies for efficiently combating colon cancer. Graphical
研究领域:
药理学
·
肿瘤学
·
纳米医学
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DOI
PubMed
高影响因子
COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS
·
2024
·
IF 4.9
Hui Ye,Jing Zhang,Guodong Yang,Rui Jiao,Shurui Han,Jing Lv,YuZhong Zhang,Lizhi Zhao,Qingping Xin,Ligang Lin,Xiaoli Ding,Hong Li
β-lactoglobulin (β-LG) is a significant allergen in milk. Enzymatic hydrolysis of β-LG produces peptides with low allergenicity and improved functionality. The biocatalytic membranes combine enzyme function and membrane separation in one step, but is still challenging for proteolysis. Herein, the biocatalytic membranes embedded with trypsin-inorganic hybrid nanoflowers are developed by a facile and green method for β-LG hydrolysis. Ethylene vinyl alcohol copolymer (EVAL) nanofibers are fabricated as 3D scaffolding on the membrane surface and trypsin-copper phosphate nanoflowers are in-situ grown in the porous structure of membranes. The nanoflowers are uniformly entrapped in the EVAL nanofiber layer and preferentially grow in the EVAL nanofibers layer, leading to the hierarchical structure of membranes. The trypsin loading of the nanoflowers membrane is approximate to 282.1 µg/cm 2 . Lower Michaelis-Menten constants Km prove the better affinity of Cu 3 (PO 4 ) 2 -TR nanoflower membrane with substrates during filtration than that in static mode, even than free trypsin. The hierarchical structure of the membranes ensures both the enhanced trypsin-substrate contacts and low mass transfer resistance during the β-LG filtration. This work not only presents a novel concept of enzyme immobilized biocatalytic membranes, but also offers a sustainable solution for high-performance proteolysis.
研究领域:
食品科学
·
生物技术
·
材料科学
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DOI
高影响因子
危险品化学品经营许可证（带存储）