Trypsin Molecules Research Articles

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor.

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.

Inhibitory effect of lignin from Canna edulis Ker residues on trypsin: kinetics and molecular docking studies

Lignin extracted from Canna edulis Ker residues shows a strong inhibitory effect on α-glucosidase and a promoting effect on α-amylase. Protease activity inhibition may play a key role in disease processes, such as metastasis, tumor invasion and bacterial colonization. Hence, in the present study, the inhibitory mechanism of lignin on trypsin was examined, including the interaction type, thermodynamic parameters, structure, reaction site and molecular docking. The isolated lignin presented an inhibitory effect on trypsin activity with an IC50 value of 1.35 μmol L-1 . This inhibition was a mixed linear type with a constant Ki of 3.92 μmol L-1 . The lignin could bind with the key amino acid residue Ser195 on the active site of the trypsin molecule to inhibit its activity, and the phenolic hydroxyl group and -OH on the β-O-4 structure of the lignin molecule were the major groups bound with trypsin. These results illustrate the inhibitory effects of Canna edulis residue lignin on protease, which helps with respect to understanding the possible application of lignin in the food industry in functional foods. © 2020 Society of Chemical Industry.

Silver Nanoparticle Trypsin Corona Formation and the Impacts on Enzymatic Potency

Herein, the protein corona formation on the spherical metal nanoparticles was studied to investigate the possible effects of silver nanoparticles (AgNPs) on the protein activity and conformation. The digestion capability of trypsin was monitored on the human serum albumin (HSA) at standard enzymatic hydrolysis conditions in the absence and presence of different concentrations of AgNPs. So the ratio of enzyme:HSA, the duration and the temperature of nanoparticle treatment were evaluated. The activity of treated trypsin molecules, in the form of hard (HC) and soft corona (SC) were studied using sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE), and nano liquid chromatography electrospray (ion trap) mass spectrometry (nano LC-ESI/MS, LC-ESI/MS). In addition the characteristics of silver nanoparticles and the formation of HSA corona on the nanoparticle surface were monitored using ultra-violet/visible spectroscopy (UV-Vis), Dynamic light scattering (DLS) and fluorescence spectrophotometry. The results demonstrated that not only the corona formation but also the AgNPs/Trypsin interaction, decreases the hydrolysis potency of trypsin. Furthermore, the encountering of the AgNPs/HSA could influence both nanoparticles and HSA molecule features. Accompanied with fluorescence study, that the HSA secondary structure. Also LC-ESI/MS data revealed the most affected HSA triptics have α-helix structure.

Green synthesis of biocompatible trypsin-conjugated Ag nanocomposite with antibacterial activity

In this work, water-soluble Ag nanoparticles were prepared in aqueous solution by using trypsin as reducing and capping agent. The protein-assisted synthetic strategy eliminates the need of intermediate protecting and linking agents compared with organometallic approach, which is simple, effect, less energy consuming, and closer to the requirements of green chemistry. The morphology, size and antibacterial activity properties could be controlled by varying experimental conditions. The results of FT-IR and SDS-PAGE analysis indicated that trypsin molecules could control the nucleation and growth of nanocrystals through chemical interaction between Ag and functional groups of trypsin. The binding of trypsin on the surface of Ag nanoparticles significantly reduced nano-toxicity through capping effect. The trypsin-conjugated Ag nanoparticles exhibited strong antibacterial activity toward both Gram-positive and Gram-negative bacteria due to small size and specific morphologies. Compared with traditional antibacterial materials, the water-solubility and biocompatibility make the products more suitable for the application in biological and medical science.

Characterization of a Kunitz trypsin inhibitor from Enterolobium timbouva with activity against Candida species

Scientific advances have not been sufficient to accompany the growing resistance to antimicrobial medicines. High mortality rates due to opportunistic infections have threated human health. The development of new drugs, such as those obtained from plant sources, is a world priority. Herein, we report the purification of a trypsin inhibitor from Enterolobium timbouva seeds (EtTI) with regard to its homology, physico-chemical and inhibitory properties. Furthermore, we evaluated its activity against Candida strains, opportunistic pathogens regularly found in hospital infections. EtTI belongs to the Kunitz family and inhibits two trypsin molecules simultaneously; a feature shared among double-headed Kunitz inhibitors. A high inhibitory activity against trypsin was determined (Ki = 0.5 nM), and refractory to digestion by pepsin. EtTI was candidicidal against C. albicans, C. buinensis and C. tropicalis, triggering disturbances on integrity of the plasma membrane and morphological alterations, presumably mediated via apoptosis. The presence of two reactive sites is an unusual feature detected in EtTI. Numerous diseases and pathologies involve changes in peptidase activities, encouraging studies with multifunctional inhibitors. Accordingly, the further exploration of EtTI could provide new insights into the Kunitz inhibitors and their applications in disease control.

Sabellastarte magnifica Carboxypeptidase Inhibitor: The first Kunitz inhibitor simultaneously interacting with carboxypeptidases and serine proteases

Multi-domain inhibitors capable to block the activity of different classes of proteases are not very common in nature. However, these kinds of molecules are attractive systems for biomedical or biotechnological applications, where two or more different targets need to be neutralized. SmCI, the Sabellastarte magnifica Carboxypeptidase Inhibitor, is a tri-domain BPTI-Kunitz inhibitor capable to inhibit serine proteases and A-like metallocarboxypeptidases. The BPTI-Kunitz family of proteins includes voltage gated channel blockers and inhibitors of serine proteases. SmCI is therefore, the only BPTI-Kunitz protein capable of inhibiting metallocarboxypeptidases. The X-ray structure of the SmCI-carboxypeptidase A complex previously obtained by us, revealed that this enzyme interacts with SmCI N-tail. In the complex, the reactive loops for serine protease inhibition remain fully exposed to the solvent in each domain, suggesting SmCI can simultaneously interact with multiple serine proteases. The twofold goals of this study were: i) to establish serine proteases-SmCI binding stoichiometry, given that the inhibitor is comprised of three potential binding domains; and ii) to determine whether or not SmCI can simultaneously bind both classes of enzymes, to which it binds individually. Our experimental approach included a variety of techniques for the study of protein-protein interactions, using as model enzymes pancreatic trypsin, elastase and carboxypeptidase A. In particular, we combined information obtained from gel filtration chromatography, denaturing electrophoresis, nuclear magnetic resonance spectroscopy and enzyme inhibition assays. Our results show that SmCI is able to bind three trypsin molecules under saturating conditions, but only one elastase interacts with the inhibitor. Additionally, we demonstrated that SmCI can bind serine proteases and carboxypeptidases at the same time (at least in the ratio 1:1:1), becoming the first protease inhibitor that simultaneously blocks these two mechanistic classes of enzymes.

Fabrication of uniform enzyme-immobilized carbohydrate microparticles with high enzymatic activity and stability via spray drying and spray freeze drying

Enzyme-immobilized particles with high enzymatic activities are fundamentally and practically important for many areas, such as pharmaceuticals, medicine, and biocatalysis. In this study, by selecting trypsin and trehalose as the representative enzyme and excipient, two techniques, spray drying (SD) and spray freeze drying (SFD), have been utilized to generate enzyme-immobilized particles and comparatively studied. In both methods, uniform enzyme-immobilized microparticles are successfully obtained by using a micro-fluidic aerosol nozzle (MFAN) as the monodisperse droplet generator. The particle morphology, size and inner structure are distinctly different between the SD- and SFD-derived trypsin/trehalose composite microparticles. The former shows crumpled morphology, smaller sizes and dense inner structure while the latter shows spherical and open porous morphology with larger particle sizes. The particle formation processes in both methods are discussed. The more surface-active and large-sized trypsin molecules tend to be accumulated at the air-liquid interface of drying droplets, leading to particle buckling in SD and the formation of thin surface trypsin-enriched layer in SFD. The trypsin enzymatic activity is highly related to the presence of trehalose and the processing method. For the pure trypsin microparticles, SFD leads to a better activity preservation than SD does due to the much higher temperature adopted in SD. The presence of trehalose can significantly protect the enzymatic activity of trypsin, reaching 97.7 ± 2.6% and 97.3 ± 1.6% activity preservation with the optimal trypsin/trehalose mass ratio of 1:1 for both the SD- and SFD-derived microparticles, respectively. The protection of the enzymatic activity originates from the hydrogen bonding formation between trypsin and trehalose and the formation of highly amorphous glass matrices, which decrease enzyme unfolding and aggregation. In terms of process operability, SD offers a rather simple and economic means to produce enzymatic microparticles of high activity with the appropriate dosage of trehalose.

In Situ Caging of Biomolecules in Graphene Hybrids for Light Modulated Bioactivity.

Remote and noninvasive modulation of protein activity is essential for applications in biotechnology and medicine. Optical control has emerged as the most attractive approach owing to its high spatial and temporal resolutions; however, it is challenging to engineer light responsive proteins. In this work, a near-infrared (NIR) light-responsive graphene-silica-trypsin (GST) nanoreactor is developed for modulating the bioactivity of trypsin molecules. Biomolecules are spatially confined and protected in the rationally designed compartment architecture, which not only reduces the possible interference but also boosts the bioreaction efficiency. Upon NIR irradiation, the photothermal effect of the GST nanoreactor enables the ultrafast in situ heating for remote activation and tuning of the bioactivity. We apply the GST nanoreactor for remote and ultrafast proteolysis of proteins, which remarkably enhances the proteolysis efficiency and reduces the bioreaction time from the overnight of using free trypsin to seconds. We envision that this work not only provides a promising tool of ultrafast and remotely controllable proteolysis for in vivo proteomics in study of tissue microenvironment and other biomedical applications but also paves the way for exploring smart artificial nanoreactors in biomolecular modulation to gain insight in dynamic biological transformation.

Core-Shell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum.

We describe the application of a fluorescently labeled water-soluble core-shell molecularly imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect trypsin. p-Aminobenzamidine (PAB), a competitive inhibitor of trypsin, was immobilized in the wells of a microtiter plate enabling the capture of trypsin in an oriented position, thus maintaining its native conformation. Fluorescent MIP nanoparticles, which bound selectively to trypsin, were used for quantification. The MIP was prepared by a multistep solid-phase synthesis approach on glass beads functionalized with PAB, orientating all trypsin molecules in the same way. The core-MIP was first synthesized, using a thermoresponsive polymer based on N-isopropylacrylamide, so as to enable its facile liberation from the immobilized template by a simple temperature change. The shell, mainly composed of allylamine to introduce primary amino groups for postconjugation of fluorescein isothiocyanate (FITC), was grafted in situ on the core-MIP, whose binding cavities were still bound and protected by the immobilized trypsin. The resulting core-shell MIP was endowed with a homogeneous population of high-affinity binding sites, all having the same orientation. The MIP has no or little cross-reactivity with other serine proteases and unrelated proteins. Our MIP-based FIA system was successfully applied to detect low trypsin concentrations spiked into nondiluted human serum with a low limit of quantification of 50 pM, which indicates the significant potential of this assay for analytical and biomedical diagnosis applications.

Covalent immobilization of trypsin onto thermo‐sensitive poly(N‐isopropylacrylamide‐co‐acrylic acid) microspheres with high activity and stability

ABSTRACTPoly(N‐isopropylacrylamide‐co‐acrylic acid) (P(NIPAM‐co‐AA)) microspheres with a high copolymerized AA content were fabricated using rapid membrane emulsification technique. The uniform size, good hydrophilicity, and thermo sensitivity of the microspheres were favorable for trypsin immobilization. Trypsin molecules were immobilized onto the microspheres surfaces by covalent attachment. The effects of various parameters such as immobilization pH value, enzyme concentration, concentration of buffer solution, and immobilization time on protein loading amount and enzyme activity were systematically investigated. Under the optimum conditions, the protein loading was 493 ± 20 mg g−1 and the activity yield of immobilized trypsin was 155% ± 3%. The maximum activity (Vmax) and Michaelis constant (Km) of immobilized enzyme were found to be 0.74 μM s−1 and 0.54 mM, respectively. The immobilized trypsin showed better thermal and storage stability than the free trypsin. The enzyme‐immobilized microspheres with high protein loading amount still can show a thermo reversible phase transition behavior. The research could provide a strategy to immobilize enzyme for application in proteomics. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43343.

Inactivation of immobilized trypsin under dissimilar conditions produces trypsin molecules with different structures

Bovine trypsin immobilized on glyoxyl agarose and submitted to different inactivation conditions produce different conformations.

Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers

Protein digestion, using an enzyme called trypsin (TR), is one of the key steps in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and not-automated due to the poor stability and autolysis of trypsin. To improve the protein digestion process, trypsin was immobilized and stabilized on polymer nanofibers entrapping superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs could be effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area for trypsin immobilization via the approach of enzyme coatings. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs; EC-TR), fabricated via simple attachment of crosslinked trypsin molecules onto NP-NFs, were highly stable and could be recycled via facile magnetic separation. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking (200rpm) for 80days, while the control samples of covalently-attached trypsin on NP-NFs (CA-TR/NP-NFs; CA-TR) and free trypsin lost more than 90% of their initial activities within 11 and 6days, respectively. When highly-stable and magnetically-separable EC-TR was employed for the repetitive digestions of enolase under recycled uses for the duration of 50days and even after treatment with another protease (chymotrypsin) for 32h, the performance of enolase digestion was successfully maintained. The use of EC-TR for the enolase digestion in the ultra-sonication system resulted in fast (∼10min) and efficient digestions with reproducible performance under recycled uses.

Characterization of the Interaction Between Pancreatic Trypsin and an Enteric Copolymer as a Tool for Several Biotechnological Applications

Protein-polyelectrolyte complexes are very interesting systems since they can be applied in many long-established and emerging areas of biotechnology. From nanotechnology to industrial processing, these complexes are used for many purposes: to build multilayer particles for biosensors; to entrap and deliver proteins for pharmaceutical applications; to isolate and immobilize proteins. The enteric copolymer poly(methacrylic acid-co-methyl methacrylate) 1:2 (MMA) has been designed for drug delivery although its chemical properties allow to use it for other applications. Understanding the interaction between trypsin and this polymer is very important in order to optimize the mechanism of formation of this complex for different biotechnological applications.The formation of the trypsin-MMA complex was studied by spectroscopy and isothermal titration calorimetry. Structural analysis of trypsin was carried out by catalytic activity assays, circular dichroism and differential scanning calorimetry. Isothermal titration calorimetry experiments showed that the insoluble complex contains 12 trypsin molecules per MMA molecule at pH 5 and they interact with high affinity to form insoluble complexes. Both electrostatic and hydrophobic forces are involved in the formation of the complex. The structure of trypsin is not affected by the presence of MMA, although it interacts with some domains of trypsin affecting its thermal denaturation as seen in the differential scanning calorimetry experiments. Its catalytic activity is not altered. Dynamic light scattering demonstrated the presence of a soluble trypsin-copolymer complex at pH 5 and 8. Turbidimetric assays show that the insoluble complex can be dissolved by low ionic strength and/or pH in order to obtain free native trypsin.

Conjugation of d-glucosamine to bovine trypsin increases thermal stability and alters functional properties

d-glucosamine was conjugated to bovine trypsin by carbodiimide chemistry, involving a water-soluble carbodiimide and a succinimide ester, with the latter being to increase the yield of the conjugation. Mass spectrometric data suggested that several glycoforms were formed, with around 12 d-glucosamine moieties coupled to each trypsin molecule on average. The moieties were probably coupled to eight carboxyl groups (of glutamyl and aspartyl residues) and to four tyrosyl residues on the surface of the enzyme. The glycated trypsin possessed increased thermal stability. When compared with its unmodified counterpart, T50% was increased by 7°C, thermal inactivation of the first step was increased 34%, and long-term stability assay revealed 71-times higher residual activity at 25°C (without stabilizing Ca2+ ions in aqueous buffer) after 67 days. Furthermore, resistance against autolysis was increased almost two-fold. Altered functional properties of the glycated trypsin were also observed. The glycated trypsin was found to become increasingly basophilic, and was found to be slightly structurally altered. This was indicated by 1.2 times higher catalytic efficiency (kcat/Km) than unmodified trypsin against the substrate N-α-benzoyl-l-arginine-p-nitroanilide. Circular dichroism spectropolarimetry suggested a minor change in spatial arrangement of α-helix/helices, resulting in an increased affinity of the glycated trypsin for this small synthetic substrate.

Poly(L-lactide) nanofibers containing trypsin for gelatin digestion

In this work, trypsin was incorporated into poly(L-lactide) (PLLA) using electrospinning technology to obtain the bioactive nanofibers for gelatin digestion. In order to improve the stability and the efficiency of trypsin incorporated PLLA nanofibers (NF-TR), glutaraldehyde was used to crosslink trypsin molecules onto fibers surface (CL-TR). CL-TR showed better gelatin digestion efficiency than NF-TR in rigorous conditions, such as at high temperatures of 40 ℃ and 50 ℃, in the presence of alcohol or dimethyl-sulfoxide, and at various pH values. CL-TR also exhibited higher stability than NF-TR. The improved efficiency and stability of CL-TR can be explained by the crosslinked trypsin on the surface of PLLA nanofibers effectively inhibited the denaturation, autolysis, and leaching of trypsin. The LC-MS/MS results showed that gelatin sample would be digested into amino acids and/or oligomers. The trypsin incorporation approach is anticipated to be employed not only for protein digestion but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.

Correlating enzyme density, conformation and activity on nanoparticle surfaces in highly functional bio-nanocomposites.

The biological activity of the immobilized enzyme is crucial for the performance of different nanoparticle mediated enzymatic assays, where enzymatic conversion can be used for label-free analyte detection. In this article we have addressed two significant aspects of enzyme-nanoparticle interactions. First, we have developed copper sulfide (CuS) nanoparticles with an average diameter of 25 nm as a potential enzyme-interface using trypsin protease as a model enzyme. CuS nanoparticles showed high trypsin immobilization capacity of about 14.0 mg m(-2) with the significant retention of native enzymatic activity (75-98%) at room temperature, even beyond the calculated tightly packed monolayer coverage (which is around 4.1 mg m(-2)). Second, we report a quantitative correlation between the structure-functional relationship and the density of immobilized trypsin on a nanoparticle surface. The in situ conformation of immobilized trypsin could be efficiently analyzed by fluorescence, circular dichroism and FT-IR spectroscopic measurements because of the small size of the nanoparticles. Trypsin molecules appear to retain their close-native tertiary and secondary structural features (with a small loss of 1-2% of helical content) in the entire surface density range (2.0-14.0 mg m(-2)) on the CuS nanoparticles. However, interestingly, at a low surface coverage (2.0 mg m(-2)), immobilized trypsin retains almost 98% of its native enzymatic activity, leading to a highly functional bio-nanocomposite. However, at higher surface coverages, the enzyme activity decreases to 77%, indicating the influence of steric crowding. Furthermore, the high functionality of the immobilized trypsin at low surface density on CuS nanoparticle was also confirmed by determining the kinetic parameters of enzymatic activity.

Potential toxicity of phthalic acid esters plasticizer: interaction of dimethyl phthalate with trypsin in vitro.

Dimethyl phthalate (DMP) is widely used as a plasticizer in industrial processes and has been reported to possess potential toxicity to the human body. In this study, the interaction between DMP and trypsin in vitro was investigated. The results of fluorescence, UV–vis, circular dichroism, and Fourier transform infrared spectra along with cyclic voltammetric measurements indicated that the remarkable fluorescence quenching and conformational changes of trypsin resulted from the formation of a DMP–trypsin complex, which was driven mainly by hydrophobic interactions. The molecular docking and trypsin activity assay showed that DMP primarily interacted with the catalytic triad of trypsin and led to the inhibition of trypsin activity. The dimensions of the individual trypsin molecules were found to become larger after binding with DMP by atomic force microscopy imaging. This study offers a comprehensive picture of DMP–trypsin interaction, which is expected to provide insights into the toxicological effect of DMP.

Protein surface charge of trypsinogen changes its activation pattern.

BackgroundTrypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction.ResultsBased on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme.ConclusionThe variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0109-5) contains supplementary material, which is available to authorized users.

EFFICIENCY OF A HIERARCHICAL DOCKING PROTOCOL FOR COMPUTATIONAL LIGAND SCREENING AGAINST HOMOLOGY MODELS

We assessed the efficiency of a hierarchical docking protocol against homology models in virtual ligand screening (VLS) studies. A low resolution model of factor X (FX) was built on a template of Trypsin molecule (PDB ID:1EB2). Afterward VLS was performed involving a hierarchical protocol, rigid body followed by flexible docking, both against model as well as an X-ray structure of FX (PDB ID:1FJS) using a smart library of 50,000 chemical compounds seeded with 9 known inhibitors of FX. The percentage enrichments of screened chemical compounds obtained both from the crystal structure and homology model of FX were compared to analyze the efficiency of the protocol. In the first 5% of the finally ranked database of the screened compounds, both against model and the X-ray structure, 67% of the inhibitors were retrieved.

A Trypsin Immobilized Sol-Gel for Protein Indentification in MALDI-MS Applications

The proteolytic enzyme trypsin was chemically immobilized to an amine-functionalized sol-gel using adipoyl chloride under nonaqueous conditions and a nitrogen atmosphere. In the synthesis of the sol-gel, tetraethyl orthosilicate (TEOS), and 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) (50:50, v/v) were used, which provided convenient physical and chemical conditions to maintain catalytic activity of immobilized trypsin molecules for the digestion of proteins in proteomics applications. Bovine serum albumin was used as a model protein to perform enzymatic digestion using the trypsin immobilized sol-gel. The resulting peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry to evaluate the digestion performance and specificity of the sol-gel material. The trypsin immobilized sol-gel showed superior enzymatic activity in protein digestion and it was determined that the sol-gel material could be repeatedly used at least 25 times without significant activity loss in long-term use. Additionally, autocatalysis was prevented by immobilization of trypsin. The peptide digest having the highest purity was obtained for protein identification studies.